The JI Acurri Cytometers
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Steel, C.
Right arrow Articles by Nutman, T. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Steel, C.
Right arrow Articles by Nutman, T. B.
The Journal of Immunology, 2003, 170: 1930-1938.
Copyright © 2003 by The American Association of Immunologists

CTLA-4 in Filarial Infections: Implications for a Role in Diminished T Cell Reactivity

Cathy Steel1 and Thomas B. Nutman

Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To determine the role that CTLA-4 might play in mediating the diminished parasite Ag-specific T cell responsiveness that is characteristically seen in filaria-infected patients, several study populations and methods were used. First, quantitative assessment of mRNA expression determined that PBMC from uninfected adolescents exposed in utero to microfilarial (Mf) Ag demonstrated a strong up-regulation of CTLA-4 to the Mf stage of the parasite in contrast to that observed in cells from children born of uninfected mothers (p = 0.005). Next, the frequency of CTLA-4 expression was examined using flow cytometry in cells from filaria-infected and -uninfected individuals ex vivo. Individuals born in filarial endemic regions of the world (with long-standing infections) had greater percentages of CD4+CTLA-4+ cells than did expatriate infected or uninfected individuals (p = 0.005 and 0.05, respectively); in addition, Mf+ patients demonstrated higher frequencies of CD4+CTLA-4+ and CD8+CTLA-4+ cells (p = 0.027 and 0.037, respectively) than did Mf- infected individuals. Of interest, the greatest intensity of CTLA-4 expression occurred in CD4+CD25+ cells, a population purported to include suppressor cells. Finally, in vitro blocking of CTLA-4 expression in PBMC from filaria-infected individuals induced a mean increase of 44% in IL-5 production to Mf Ag, whereas there was a concurrent mean decrease of 42% in IFN-{gamma} production, suggesting that CTLA-4 also acts to alter the Th1/Th2 balance in filaria-infected individuals. Together, these data indicate a significant role for CTLA-4 in regulating the host response to filarial infections and that factors such as length of exposure and patency are important codeterminants.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Filarial infections, caused by vector-borne helminth parasites, characteristically are associated with a profound down-regulated T cell response to parasite Ag in infected individuals when contrasted to filaria-exposed, but uninfected, individuals (1, 2, 3). Among the many theories used to explain the modulated parasite Ag-specific cellular responses (particularly T cell proliferation and cytokine production) seen in patients with patent filarial infections, those relating to regulatory factors and/or cells have been predominant. In particular, both down-regulation by IL-10 and/or TGF-{beta} (4, 5, 6, 7), regulation by Th3 or Tr1 cells (3), and changes in the balance of type 1 and type 2 CD4+ cells (4, 8) have each been implicated as important mechanisms underlying the filaria-specific down-regulated state. Indeed, >2 decades ago it was demonstrated that patently infected (microfilaria-positive (Mf+)2) patients had demonstrable Ag-specific suppressor T cells (9) as well as non-T cell-derived suppressor factors (10). More recently, it has been shown that most, but not all, (11) of this modulating activity is a consequence of the interaction between the microfilarial stage of the parasite (12) and the host cellular immune system, some of which may occur neonatally (13, 14, 15, 16, 17, 18).

Although not well studied in chronic parasitic infections, another possible pathway in the suppression of parasite-specific responses could be through the engagement of the T cell costimulatory molecule CTLA-4. CTLA-4, which is stored in endosomal compartments and expressed on the cell surface following activation (19), delivers an inhibitory signal to T cells in contrast to its homologue CD28. It is generally believed that CTLA-4 functions by inhibiting IL-2 (20, 21, 22) and cell cycle progression (22, 23, 24), but it has also been shown to mediate Ag-specific apoptosis (25). Binding of CTLA-4 to its ligands (CD80 and CD86) occurs with a higher affinity than occurs with CD28 (19); therefore, depending on the level of ligand expression (26) and the strength of TCR signaling (27, 28), this can alter the nature of the immune response. Indeed, it has been demonstrated that blocking the binding of CTLA-4 to its ligands can increase type 2 CD4+ cytokine secretion (29, 30).

The role of CTLA-4 in parasitic infections has recently been explored in mouse models (31, 32) and in some human studies (33). In this study we have begun to assess a possible role for this molecule in the T cell hyporesponsiveness observed in filaria-infected patients. Our results indicate that CTLA-4 does contribute to the Ag-specific down-regulation of cellular responsiveness seen in filaria-infected patients and points to the importance of such factors as length of exposure, microfilarial status, and in utero contact with parasite Ag. In addition, we show that interfering with CTLA-4 may alter the Th1/Th2 balance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

The parasite Ag used in this study were extracts of adult (BmA), microfilarial (Mf Ag), and L3 stages of the parasite Brugia malayi (34, 35). Previous work in our laboratory has demonstrated extensive cross-reactivity among filarial species, and therefore B. malayi was used because of its greater availability. All Ag preparations were below the detectable levels of endotoxin by the Limulus amebocyte lysate assay (QCL-1000 kit; BioWhittaker, Walkersville, MD). Nonparasite Ag were streptolysin O (SLO; Difco, Detroit, MI) and tuberculin purified protein derivative (PPD; Connaught, Ontario, Canada).

Ab used for surface and intracellular flow cytometry were obtained from BD PharMingen/BD Biosciences (San Diego, CA) unless otherwise noted. Ab included PE-conjugated CTLA-4 (CD152) and CD28-PE, FITC-conjugated CD25, and PerCP-conjugated CD4 and CD8. Control Ab were mouse IgG1/PE (Caltag, Burlingame, CA), mouse IgG1-FITC (Caltag), mouse IgG2a-PE, and mouse IgG1-PerCP.

Study populations

Several populations were used for this study, as shown in Table I. For assessment of mRNA expression of costimulatory molecules, PBMC were obtained from 21 young adults from the Wuchereria bancrofti-endemic island of Mauke in the Cook Islands. These individuals were determined to be uninfected both by blood filtration and by testing for circulating filaria Ag and differed from each other only on the basis of maternal infection status during their pregnancy, as described previously (13). For the remaining studies, filaria-infected (n = 66) or -uninfected (n = 23) individuals seen at the National Institutes of Health were used. Of the 66 infected individuals, 17 were recent immigrants to the U.S. from endemic regions of the world, and 49 were North American expatriates who had been temporary residents of filaria-endemic regions. Among the patients with filarial infections, 41 had loaisis (Loa loa), 8 had lymphatic filariasis (W. bancrofti), and 14 had onchocerciasis (Onchocerca volvulus); three patients had mixed infections. In addition, a subset (n = 15) of these patients was re-examined following curative treatment. Not all patients were used for each analysis; the numbers used are noted in the text and/or figures as appropriate.


View this table:
[in this window]
[in a new window]
  		Table I. Study populationsa

 
Semiquantitative detection of costimulatory molecules in patient cells by RT-PCR

To quantify the amounts of CTLA-4, CD28, CD80, and CD86 mRNA in PBMC, RNA was obtained from PBMC (2 x 106 cells) cultured in the presence or the absence of parasite (BmA (5 µg/ml), Mf (1 µg/ml), and L3 (0.25 µg/ml)) or nonparasite (PPD (10 µg/ml) and SLO (1/100 final concentration)) Ag for 12, 48, or 72 h (13) using RNAstat (Tel-Test, Friendswood, TX). Total RNA (1 µg) was reversed transcribed using oligo(dT) to prime the reaction, following which PCR was performed using one biotinylated and one unbiotinylated primer specific for each of the costimulatory molecules. Detection and quantification of the PCR products were performed using an ELISA-based system as described previously (36) that employed streptavidin-coated microtiter plates, a fluorescein-labeled probe, alkaline phosphatase-labeled anti-fluorescein Ab, and an ELISA amplification system (Invitrogen, Carlsbad, CA) as the substrate. To provide quantification, OD readings for the costimulatory molecules and corresponding hypoxanthine-guanine phosphoribosyltransferase (HPRT) were compared using a standardized curve comprising cDNA made from PMA/ionomycin-driven PBMC run in each assay. Values for each of the costimulatory molecules were normalized to the HPRT values from the same samples. For illustration of the data, the media background (also normalized to HPRT) was subtracted. The sequences of the primers and probes used were as follows: CTLA-4, 5'-TGA GGT CCG GGT GAC AGT GCT-3', 5'-biotin-CCG TTG CCT ATG CCC AGG TAG-3', and 5'-fluorescein (Flu)-CAC TAT CCA AGG ACT GAG GGC-3'; CD28, 5'-TGC GTC TTT CAG TTC CCC TC-3', 5'-biotin-CCC AAT TTC CCA TCA CAG TTG-3', and 5'-Flu-GCA GTC GCC CAT GCT TGT AG-3'; CD80, 5'-AGC AAG CTG TGA AAC TAA ATC CAC AAC CTT-3', 5'biotin-AAC AAC ACA CTC GTA TGT GCC CTC GTC AGA-3', and 5'-Flu-GGC CAC ACA CGG AGG CAG G-3'; CD86, 5'-CAC AGC AGA AGC AGC CAA AAT GG-3', 5'-biotin-GCC CTT GTC CTT GAT CTG AAG CAT TG-3', and 5'-Flu-CCA TGC CAA TTT GCA AAC TC-3'; and HPRT, 5'-CGA GAT GTG ATG AAG GAG ATG G-3', 5'-biotin-GGA TTA TAC TGC CTG ACC AAG G-5', and 5'-Flu-TGG TGG AGA TGA TCT CTC AA-3'.

Ex vivo detection of CTLA-4 and CD28

Cryopreserverd PBMC from patients with various filarial infections were incubated for 24 h in RPMI (BioWhittaker) supplemented with 10% FCS (Atlanta Biological, Norcross, GA), glutamine, and gentamicin (C-RPMI/10% FCS) at 37°C in 5% CO2. After 24 h cells were fixed in 4% paraformaldehyde and permeabilized in PBS/0.1% saponin for intracellular detection of CTLA-4. Staining of CD4 and CD8 was performed concurrently. In separate experiments cells were stained first for surface expression of CD4 and CD25, followed by fixation and permeabilization for staining with {alpha}CTLA-4; other cells were stained solely for surface expression of CD4, CD8, and CD28. Fluorescence was measured on a FACSCalibur (BD Biosciences, San Jose, CA) using gated lymphocytes.

In vitro blocking of CTLA-4

To test the effect of blocking CTLA-4 (but not its homologue CD28) on PBMC, Fab of {alpha}CTLA-4 and its control Ab, mouse IgG2a (BD PharMingen), were made using the ImmunoPure Fab preparation kit (Pierce, Rockford, IL) following the manufacturer’s instructions. SDS-PAGE was used to confirm the effectiveness of the papain digestion. PBMC from eight filaria-infected patients were cultured at 1 x 106 cells/ml in C-RPMI/10% FCS in 48-well plates (Costar, Cambridge, MA) at 37°C in 5% CO2. Cells were cultured in the absence (medium alone) or the presence of either Mf Ag (5 µg/ml), PPD (10 µg/ml), or SLO (1/100 final concentration) with or without {alpha}CTLA-4 Fab or its control Fab Ab (both at 20 µg/ml). Culture supernatants were collected at 5 days for measurement of IFN-{gamma} and IL-5 by ELISA using the Ab pairs TRFK5 and 5A10 (BD PharMingen) for IL-5, and M700A and M-701-B (Endogen, Boston, MA) for IFN-{gamma}. OD were read at 405 nm using standardized curves based on recombinant human cytokines (recombinant human IL-5 (BD PharMingen) and recombinant human IFN-{gamma} (InterMune, Brisbane, CA)). The limits of detection were 15 pg/ml for IL-5 and 39 pg/ml for IFN-{gamma}.

In some experiments cells were cultured at 100,000 cells/well in 0.2 ml in a 96-well plate (Costar) using the same culture conditions. On day 5, cells were pulsed with 1 µCi/well [3H]thymidine and harvested 18 h later for measurement of proliferation.

Statistical analyses

Comparisons between groups of unpaired data were performed using the Mann-Whitney U test. The Wilcoxon signed-rank test was used to compare paired data (i.e., pre- and post-treatment data).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of in utero exposure to filarial infection

To determine the effect of in utero exposure to parasite Ag on the expression of costimulatory molecules, semiquantitative RT-PCR was performed on cDNA from young adults with no history of infection, whose mothers were either infected with W. bancrofti and microfilaremic (n = 11) or were uninfected (n = 10) during their pregnancy. Because peak mRNA expression of costimulatory molecules occurred at 48 h, this time point was used in the analysis. Although there were no differences between the two groups in the expression of CD28, CD80, or CD86 mRNA, either spontaneously or to any of the Ag, for CTLA-4, children born to infected mothers had markedly increased expression in response to Mf Ag compared with those born to uninfected mothers (Fig. 1; median, 135 U/ml; p = 0.005 compared with children of uninfected mothers) despite the fact that spontaneous expression of CTLA-4 mRNA did not differ significantly, p = 0.099). Moreover, the expression of CTLA-4 mRNA in children born to uninfected mothers was down-regulated in response to Mf Ag compared with spontaneous production (median, -123 U/ml). CTLA-4 expression in response to other Ag, both parasitic and nonparasitic, did not differ significantly between the two groups of young adults.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 1. Semiquantitative RT-PCR assessment of 48-h mRNA expression of CTLA-4 in response to parasite and nonparasite Ag. Each box ( , children born to infected Mf+ mothers (n = 11); {square}, children born to uninfected mothers (n = 10)) representing the relative amounts of CTLA-4 (expressed as units (u) normalized to HPRT) is composed of three horizontal lines representing the 25th, 50th, and 75th percentiles of the data. Vertical lines represent the 10th and 90th percentiles, with circles denoting values outside these percentiles. The p values were calculated using the Mann-Whitney U test.

 
Ex vivo production of CTLA-4 and CD28 in filarial patients

Because children born to infected mothers had evidence of long term decreased T cell responses to Mf Ag (13) and increased CTLA-4 mRNA expression to this stage of the parasite, the role of CTLA-4 in modulating the immune response to filarial infection was examined using cells obtained from clinically well-defined, filaria-infected and -uninfected patients whose length of infection and clinical status differed. Fig. 2 illustrates spontaneous ex vivo CTLA-4 expression in CD4+ cells from filaria-infected endemic and expatriate patients compared with uninfected individuals (normal subjects). Because no differences were found among patients with different filarial infections (i.e., loiasis, onchocerciasis, lymphatic filariasis), patients with different infections were grouped together for analysis. CD4+ cells from endemic patients had, as a group, greater spontaneous CTLA-4 expression than those from either expatriate or normal individuals (GM % of cells, 31, 25, and 26%, respectively). There was no correlation between age (and presumably length of exposure) and expression of CTLA-4 in endemic individuals (data not shown), nor were there any differences in CTLA-4 expression by CD8+ cells. Of interest, cells from expatriates (those acquiring infection through travel or temporary residence in endemic areas) did not differ significantly from those from nonendemic normal subjects in their expression of CTLA-4. In addition, there were only slight variations in the mean fluorescence intensities (MFI) of CTLA-4, indicating that the primary differences seen were in the percentage of cells expressing CTLA-4 rather than in the amount expressed per cell (data not shown).



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 2. Percentage of CD4+ T lymphocytes expressing intracellular CTLA-4 for endemic and expatriate filaria-infected patients and for uninfected normal controls. Data are expressed through box plots that illustrate the 25th, 50th, and 75th percentiles (horizontal lines) of the frequencies of CD4+CTLA-4+ cells as assessed by intracellular flow cytometry. Vertical lines represent the 10th and 90th percentiles, and circles denote values outside these percentiles. The p values were calculated using the Mann-Whitney U test.

 
Mf status was also a factor in the cellular expression of CTLA-4, as Mf+ patients had a significantly higher percentage of both CD4+ (p = 0.027) and CD8+ cells (p = 0.037) positive for CTLA-4 than did Mf- patients (Fig. 3). There were no significant differences between Mf+ patients born in endemic regions (n = 12) and expatriate Mf+ patients (n = 15) in the percentage of CD4+ CTLA-4+ cells, presumably indicating that Mf status is a more important determinant for CTLA-4 expression than is length of exposure. Of interest, the cells producing the highest per cell amount of CTLA-4 for all patients were of the CD4+CD25+ phenotype (Fig. 4), a population purported to include T suppressor cells (GM MFI, 16.97 for CD4+CD25+ cells and 12.01 for CD4+CD25- cells; p < 0.0001; Fig. 4C).



View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 3. Percentage of CD4+ and CD8+ T lymphocytes expressing intracellular CTLA-4 in microfilaremic (Mf+) and amicrofilaremic (Mf-) patients. Data are expressed as box plots ( , Mf+ patients; {square}, Mf- patients) as described in Fig. 2. The p values were calculated using the Mann-Whitney U test.

 


View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 4. MFI of CTLA-4 in CD4+CD25+ cells are greater than those in CD4+CD25- cells. A, Cells were stained for coexpression of CD4 and CD25 (gate shown in red, left panel) and then examined for CTLA-4 expression (red dots, right panel). B, Histogram plot comparing the MFI of CTLA-4+ cells in the CD4+CD25+ (blue) and CD4+CD25- (red) populations. The dotted line represents the isotype control. C, Box plots (as described in Fig. 2) denoting the MFI of CTLA-4 expression in the CD4+CD25+ and CD4+CD25- populations (n = 59 patients). The p value was calculated using the Mann-Whitney U test.

 
While the major outcome of CTLA-4 engagement is the inhibition of T cell responses, its homologue CD28 (which is activated before CTLA-4) delivers an opposing signal; thus, its constitutive expression was examined for comparison. As CD28 is found to be expressed on the majority of cells (particularly CD4+ cells), differences in MFI were assessed (Fig. 5). As demonstrated, the per cell expression of CD28 on CD4+ cells was higher in cells from infected endemic patients than in those from either infected expatriate or uninfected normal individuals, with the greatest difference occurring between endemic and expatriate patients (GM MFI, 71.9 and 53.8, respectively); however, both endemic patients and uninfected normal individuals had greater CD28 expression per cell in the CD8+ population than that found in expatriates. In addition, both CD4+ and CD8+ cells in Mf+ patients were found to have higher MFI than those in Mf- individuals (GM CD4+ MFI, 64.4 and 54.1, respectively (p = 0.003); GM CD8+ MFI, 38.4 and 32.4, respectively (p = 0.037); data not shown). Notably, in contrast to what was observed for CTLA-4, the length of exposure appeared to be an important factor for determining the expression of CD28, as there was significantly higher expression of CD28 on CD4+ cells from endemic Mf+ patients compared with Mf+ expatriates (p = 0.001), with a similar trend for CD8+ cells (p = 0.057).



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 5. MFI of CD28 as expressed in CD4+ and CD8+ cells in endemic and expatriate infected individuals and in uninfected normal controls. Surface expression of CD28 as assessed by flow cytometry is illustrated by box plots ( , endemic patients; {square}, expatriate patients; , normal controls) as described in Fig. 2. The p values were calculated using the Mann-Whitney U test.

 
To assess the expression of these important T cell costimulatory molecules following treatment, a subset of patients was studied >10 years following initial treatment for their filarial infections, and these subjects were, by all available criteria, definitively cured (37). Cells obtained both pre- and post-treatment from these patients were analyzed concurrently ex vivo for the expression of CTLA-4 and for surface expression of CD28. The MFI of CD28 increased post-treatment in the CD4+ cells of 10 of 14 patients (GM MFI, 56.2 (pretreatment) and 65.0 (post-treatment); Fig. 6) and in the CD8+ cells of eight of 14 individuals (GM MFI, 32.6 (pretreatment) and 37.7 (post-treatment)). In contrast, changes in the expression of CTLA-4 by both CD4+ and CD8+ cells were not consistent, increasing in some individuals post-treatment and decreasing in others (GM % for CD4+ cells, 25.2% (pretreatment) and 25.6% (post-treatment); GM (%) for CD8+ cells, 7.2% (pretreatment) and 6.5% (post-treatment)).



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 6. Changes in the expression of CD28 and CTLA-4 in filaria-infected individuals (n = 15) pre- and 10 years post-treatment. The patient population consisted of two endemic and 13 expatriate individuals, nine of whom were Mf+ and six of whom were Mf-. A, The MFI of surface CD28 expression for CD4+ (left panel) and CD8+ (right panel) cells. B, The percentage of cells expressing intracellular CTLA-4 in CD4+ (left panel) and CD8+ (right panel) cells. Each line represents an individual patient studied before and following definitive treatment. The p values were calculated using the Wilcoxon signed-rank test.

 
Blocking CTLA-4 in vitro

To determine whether CTLA-4 could play a role in altering the responses to parasite Ag, a Fab of {alpha}CTLA-4 was used to block (without activating) CTLA-4 to examine its effect on cytokine production in response to parasite and nonparasite Ag. In preliminary studies with cells from four filaria-infected patients, the use of {alpha}CTLA-4 Fab in vitro was shown to increase Ag-specific proliferation (compared with the control Fab Ab) in patient PBMC by an average of 31 and 34% in response to Mf Ag and PPD, respectively, but did not alter the response to SLO (increase, 2.8%). To examine further the effect of blocking CTLA-4, the effect of {alpha}CTLA-4 Fab on the cytokine responses in eight patients (six endemic and two expatriate; six of eight Mf+) was examined. If CTLA-4 were playing a role in the inhibition of both Th1 and Th2 cytokines in filarial infections, an increase in both IFN-{gamma} and IL-5 in response to Mf Ag would be expected in the presence of blocking Ab; however, while neutralizing {alpha}CTLA-4 Ab significantly increased the production of IL-5 following exposure of PBMC to Mf Ag in all eight individuals (GM % increase over control Fab Ab, 44%; Fig. 7), changes in IFN-{gamma} were variable, with five of eight individuals’ PBMC demonstrating decreased production in the presence of {alpha}CTLA-4 compared with the control Ab (GM % decrease, 42%). In contrast, the production of IFN-{gamma} did increase significantly in response to PPD in the presence of blocking Ab to CTLA-4 and less so in response to SLO (GM % increases over control, 31 and 16%, respectively). As found for Mf Ag, IL-5 production also increased in the presence of PPD and {alpha}CTLA-4 Fab (GM % increase over control, 39%), but only slightly in response to SLO (GM % increase, 8%).



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 7. Blocking of CTLA-4 in vitro alters the production of IFN-{gamma} and IL-5. PBMC from eight patients (one with onchocerciasis, two with lymphatic filariasis, and five with loiasis) were cultured in the presence of parasite (Mf Ag) or nonparasite (PPD and SLO) Ag in the presence or the absence of blocking Ab to CTLA-4 for 5 days. Culture supernatants were measured for IFN-{gamma} ({circ}) and IL-5 (•). Each circle represents an individual patient. Data are expressed as a percentage of the control Ab; the shaded area represents either no change or a negative change.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The role of CTLA-4 in controlling immune responses has been well documented, particularly in animal models of autoimmunity (38, 39, 40). Its role in chronic parasitic infections, however, has been less well examined. Recently, it was demonstrated that a group of immigrants with intestinal helminths had reduced proliferation to recall Ag accompanied by an increase in CTLA-4 expression (33). This was associated with an apparent impairment of transmembrane signaling demonstrated by attenuated phosphorylation of ERK1 and ERK2 and reduced activation of I{kappa}b{alpha} and p38 following stimulation with PMA and Ca2+ ionophore.

Filarial infection is an even more chronic infection associated with more profound impairment of T cell responses that can continue even beyond clearance of the parasite (41, 42). Significant pathologic reactions can occur in a subset of patients chronically infected with filarial parasites, among them chronic lymphatic obstruction and elephantiasis (seen in lymphatic filariasis) and debilitating skin and eye inflammation (seen in onchocerciasis). These sequelae may arise as a consequence of the patient’s inflammatory reactions to the parasite or parasite products. In many cross-sectional studies, individuals with pathology may demonstrate greater T cell reactivity in vitro to parasite Ag than patients who are clinically asymptomatic (12). Similarly, infected expatriates (43) and infected individuals from areas where the filariae have only recently been introduced (44), who have, by definition, shorter exposure time than patients with long-standing chronic infection, also have stronger T cell responses to filarial Ag. The differences in responsiveness may depend on the state of T cell activation (28). The data from the present study suggest that CTLA-4 plays a significant role in these differences. Indeed, our experiments demonstrate that both the length of exposure and Mf status are important factors in determining the level of CTLA-4 expression and may provide insight into the T cell hyporesponsiveness seen in Mf+ individuals.

Length of exposure and Mf status were also determinants in the relative amount of CD28 expressed per cell (see Fig. 5). Of interest, T cells from chronically exposed individuals with life-long exposure to the parasite (so-called endemic patients), despite their increase in CTLA-4, also showed greater expression of CD28. This suggests perhaps that there is a compensatory increase in CD28 expression under chronic exposure to parasite Ag to counterbalance the down-regulatory effects of CTLA-4. With chronic antigenic exposure, the ratio of Ag to MHC is increased, presumably allowing CD28 to compete with CTLA-4 (which typically has a stronger affinity for the ligands CD80 and CD86); however, because CTLA-4 is activated subsequent to CD28 and is also expressed on the cell surface subsequently, the immune response to the parasite might continue in an anergized state, as an increase in CD28 expression appears to be followed by an increase in CTLA-4 expression.

The mechanisms by which CTLA-4 functions in filarial infections are probably multiple. CTLA-4 may operate, in a manner similar to that seen in the prevention of autoimmune and lymphoproliferative disorders, by down-regulating those inflammatory responses that can cause debilitating disease. Alternatively, it may be through the interaction of IL-10, a cytokine that, when available to dendritic cells, induces a population of anergic T cells that express high levels of both intracellular and extracellular CTLA-4. As a consequence, autologous T cells are suppressed, a process that is reversible by blocking CTLA-4 function (45). In filarial infections, IL-10 has been implicated in the down-regulation of parasite-specific responses (5, 44, 46). Indeed, cells from asymptomatic Mf+ patients have been shown to produce IL-10 spontaneously in greater amounts than those patients with symptomatic infection (i.e., elephantiasis) (2, 5). Characteristically, APC are a major source of this cytokine, a finding given credence by studies of filarial infections in mouse models (6). Further, it is the Ag derived from the Mf stage of the parasite that induces this important down-regulatory cytokine (12).

It is also to this Mf stage that long term T cell anergy was induced in children born to filaria-infected Mf+ mothers (13). These same children, as demonstrated in the present study, showed increased mRNA expression of CTLA-4 in response to Mf Ag, but not to the other parasite stages tested. Early in utero exposure to this stage of the parasite is more than likely responsible for the tolerance seen in many infected individuals from endemic areas. One explanation for the tolerance seen could be the effect of specific Mf Ag on an immature immune system (i.e., one that has immature APC with low expression of CD80 and CD86). Indeed, it has been shown that low levels of CD86 can partially inhibit T cell proliferation mediated by interactions with CTLA-4 (26), and Mf+ patients have been found to express less CD80 and CD86 than both endemic uninfected individuals and patients with elephantiasis (47). In addition, low densities of agonist peptide-MHC complexes, which may be a factor in in utero exposure to filarial Ag, can result in memory CD4+ T cell anergy, an anergy that, in other systems, can be reversed with blocking Ab to CTLA-4 (48). Further, under normal conditions, the majority of fetal T cells are naive (CD45RA+) and most likely express little, if any, CTLA-4. Chronic in utero exposure to parasite Ag may alter both the memory/naive T cell ratio and increase the expression of CTLA-4 as more cells become activated. The fact that CTLA-4 expression continues to increase in response to Mf Ag even many years later in uninfected children born to Mf+ mothers suggests that those changes experienced in utero may continue later in life if the individual is under constant environmental exposure to the parasites or parasite Ag (i.e., exposure to insect vectors with the infected larval stage).

While direct down-regulation of T cell responses may be one component of CTLA-4 function, another result of its actions appears to be a shift in the Th1/Th2 balance. In two studies using mouse models, amelioration of disease caused by the intracellular parasite Leishmania major (31) and the intestinal helminth Nippostrongylus brasiliensis (32) occurred with blockade of CTLA-4, principally due to a shift in the Th1 and Th2 balance. Studies using CTLA-4 knockout mice have demonstrated that these mice produce high levels of the Th2 cytokines (IL-4 and IL-5) and, in some cases, show altered responses to {alpha}CD28 costimulation (24, 29, 30). In addition, engagement of CTLA-4 in mice suppresses IL-4 production and leads to the development of Th1 cells, while blockade of CTLA-4 polarizes naive cells to a Th2 phenotype (49). Human studies, as well, have shown that Th2 clones express higher percentages of CTLA-4 (50) and that blockade of CTLA-4 in vitro enhances Th2 secretion (28, 51). It has been suggested that blocking CTLA-4 alters the strength of the signal delivered to a T cell, which, in turn, would allow the development of IL-4-producing cells that normally depend upon stronger stimuli (51). With regard to the Th1/Th2 dichotomy, similar findings were seen in the present study in which blocking CTLA-4 led to increased production of IL-5, but decreased secretion of IFN-{gamma} to Mf Ag. This finding, at least as seen with IFN-{gamma}, appeared to be parasite specific, as production of IFN-{gamma} increased in response to the two nonparasite Ag tested. Of interest, IL-5 (52, 53, 54, 55) and other Th2 cytokines (56, 57, 58) have been shown to be important in immunity to helminth infections in mouse models, and in addition, the percentage of patients who produced IL-5 to Mf Ag increased after the cure of their long term filarial infections (41).

The mechanism by which CTLA-4 down-regulates the production of IL-5 (and presumably other Th2 cytokines) may be indirect. Several studies have demonstrated that CTLA-4 inhibits IL-2 (20, 21, 26, 59), even at the level of transcription (22). We have previously demonstrated that endemic individuals infected with onchocerciasis produce less parasite-specific IL-5 than endemic normal subjects and that this lack of IL-5 was IL-2 dependent (60). In some preliminary results using a small subset of patients from this study, in vitro IL-2 production increased in some in response to Mf Ag in the presence of {alpha}CTLA-4 Fab (data not shown); however, more work needs to be performed to determine whether CTLA-4 is altering the production of IL-5 through IL-2.

Finally, down-regulation of filaria-specific responses may also occur through the actions of CD4+CD25+ suppressor cells. While some studies find that CTLA-4 plays a role in the suppressor activity of these CD25+ cells (40, 61), others have been unable to demonstrate such a role (62). In the present study it is intriguing that this CD4+CD25+ cell population expressed higher amounts of CTLA-4 per cell than did CD4+CD25- cells, although the role, if any, of these cells in filarial infections has yet to be determined. In addition, while it would be interesting to determine whether this cell population also produces greater IL-10 (and possibly TGF-{beta}), the spontaneous measurement of these cytokines in this small population would be difficult to evaluate, as we have found that only ~0.5–2% of all CD4 cells produce IL-10 spontaneously.

While there is no single unifying mechanism to explain the cellular hyporesponsiveness seen in filaria-infected individuals, our data indicate that CTLA-4 plays an important role. That curative treatment of filarial infections did not significantly alter the spontaneous expression of CTLA-4, while CD28 expression did increase, may reflect a higher baseline setting as a consequence of chronic exposure to filarial parasites. CTLA-4 expression in filarial infections may function to down-regulate inflammatory responses that could lead to the debilitating effects of these diseases; concurrently, however, it may also prevent the elimination of the parasite by altering cytokine profiles and modulating T cell activation. It is this balancing and counterbalancing that has evolved in this host/parasite relationship and provides insight into how these threadlike parasites use the host immune system to increase their own survival.


   Acknowledgments
 
We thank Brenda Rae Marshall for help in preparing this manuscript.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Cathy Steel, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, 4 Center Drive, Room 4/126, National Institutes of Health, Bethesda, MD 20892-0245. E-mail address: csteel{at}niaid.nih.gov Back

2 Abbreviations used in this paper: Mf, microfilarial; Flu, fluorescein; GM, geometric mean; HPRT, hypoxanthine-guanine phosphoribosyltransferase; MFI, mean fluorescence intensity; PPD, purified protein derivative; SLO, streptolysin O. Back

Received for publication August 12, 2002. Accepted for publication December 2, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Steel, C., A. Guinea, E. A. Ottesen. 1996. Evidence for protective immunity to bancroftian filariasis in the Cook Islands. J. Infect. Dis. 174:598.[Medline]
  2. Soboslay, P. T., S. M. Geiger, N. Weiss, M. Banla, C. G. Luder, C. M. Dreweck, E. Batchassi, B. A. Boatin, A. Stadler, H. Schulz-Key. 1997. The diverse expression of immunity in humans at distinct states of Onchocerca volvulus infection. Immunology 90:592.[Medline]
  3. Doetze, A., J. Satoguina, G. Burchard, T. Rau, C. Loliger, B. Fleischer, A. Hoerauf. 2000. Antigen-specific cellular hyporesponsiveness in a chronic human helminth infection is mediated by T(h)3/T(r)1-type cytokines IL-10 and transforming growth factor-{beta} but not by a T(h)1 to T(h)2 shift. Int. Immunol. 12:623.[Abstract/Free Full Text]
  4. King, C. L., S. Mahanty, V. Kumaraswami, J. S. Abrams, J. Regunathan, K. Jayaraman, E. A. Ottesen, T. B. Nutman. 1993. Cytokine control of parasite-specific anergy in human lymphatic filariasis: preferential induction of a regulatory T helper type 2 lymphocyte subset. J. Clin. Invest. 92:1667.
  5. Mahanty, S., S. N. Mollis, M. Ravichandran, J. S. Abrams, V. Kumaraswami, K. Jayaraman, E. A. Ottesen, T. B. Nutman. 1996. High levels of spontaneous and parasite antigen-driven interleukin-10 production are associated with antigen-specific hyporesponsiveness in human lymphatic filariasis. J. Infect. Dis. 173:769.[Medline]
  6. Osborne, J., E. Devaney. 1999. Interleukin-10 and antigen-presenting cells actively suppress Th1 cells in BALB/c mice infected with the filarial parasite Brugia pahangi. Infect. Immun. 67:1599.[Abstract/Free Full Text]
  7. Mahanty, S., M. Ravichandran, U. Raman, K. Jayaraman, V. Kumaraswami, T. B. Nutman. 1997. Regulation of parasite antigen-driven immune responses by interleukin-10 (IL-10) and IL-12 in lymphatic filariasis. Infect. Immun. 65:1742.[Abstract]
  8. Osborne, J., S. J. Hunter, E. Devaney, J. S. Jenson, R. O’Connor. 1996. Anti-interleukin-4 modulation of the Th2 polarized response to the parasitic nematode Brugia pahangi. Infect. Immun. 64:3461.[Abstract]
  9. Piessens, W. F., F. Partono, S. L. Hoffman, S. Ratiwayanto, P. W. Piessens, J. R. Palmieri, I. Koiman, D. T. Dennis, W. P. Carney. 1982. Antigen-specific suppressor T lymphocytes in human lymphatic filariasis. N. Engl. J. Med. 307:144.[Abstract]
  10. Piessens, W. F., S. Ratiwayanto, S. Tuti, J. H. Palmieri, P. W. Piessens, I. Koiman, D. T. Dennis. 1980. Antigen-specific suppressor cells and suppressor factors in human filariasis with Brugia malayi. N. Engl. J. Med. 302:833.[Abstract]
  11. Bosshardt, S. C., S. U. Coleman, C. S. McVay, K. L. Jones, T. R. Klei. 1995. Impact of microfilaremia on maintenance of a hyporesponsive cellular immune response in Brugia-infected gerbils (Meriones unguiculatus). Infect. Immun. 63:940.[Abstract]
  12. Mahanty, S., H. E. Luke, V. Kumaraswami, P. R. Narayanan, V. Vijayshekaran, T. B. Nutman. 1996. Stage-specific induction of cytokines regulates the immune response in lymphatic filariasis. Exp. Parasitol. 84:282.[Medline]
  13. Steel, C., A. Guinea, J. S. McCarthy, E. A. Ottesen. 1994. Long-term effect of prenatal exposure to maternal microfilaraemia on immune responsiveness to filarial parasite antigens. Lancet 343:890.[Medline]
  14. Elson, L. H., A. Days, M. Calvopina, W. Paredes, E. Araujo, R. H. Guderian, J. E. Bradley, T. B. Nutman. 1996. In utero exposure to Onchocerca volvulus: relationship to subsequent infection intensity and cellular immune responsiveness. Infect. Immun. 64:5061.[Abstract]
  15. Pit, D. S., A. M. Polderman, H. Schulz-Key, P. T. Soboslay. 2000. Prenatal immune priming with helminth infections: parasite-specific cellular reactivity and Th1 and Th2 cytokine responses in neonates. Allergy 55:732.[Medline]
  16. Lammie, P. J., W. L. Hitch, E. M. Walker Allen, W. Hightower, M. L. Eberhard. 1991. Maternal filarial infection as risk factor for infection in children. Lancet 337:1005.[Medline]
  17. Klei, T. R., D. P. Blanchard, S. U. Coleman. 1986. Development of Brugia pahangi infections and lymphatic lesions in male offspring of female jirds with homologous infections. Trans. R. Soc. Trop. Med. Hyg. 80:214.[Medline]
  18. Schrater, A. F., A. Spielman, W. F. Piessens. 1983. Predisposition to Brugia malayi microfilaremia in progeny of infected gerbils. Am. J. Trop. Med. Hyg. 32:1306.
  19. Linsley, P. S., J. L. Greene, P. Tan, J. Bradshaw, J. A. Ledbetter, C. Anasetti, N. K. Damle. 1992. Coexpression and functional cooperation of CTLA-4 and CD28 on activated T lymphocytes. J. Exp. Med. 176:1595.[Abstract/Free Full Text]
  20. Walunas, T. L., C. Y. Bakker, J. A. Bluestone. 1996. CTLA-4 ligation blocks CD28-dependent T cell activation. J. Exp. Med. 183:2541.[Abstract/Free Full Text]
  21. Blair, P. J., J. L. Riley, B. L. Levine, K. P. Lee, N. Craighead, T. Francomano, S. J. Perfetto, G. S. Gray, B. M. Carreno, C. H. June. 1998. CTLA-4 ligation delivers a unique signal to resting human CD4 T cells that inhibits interleukin-2 secretion but allows Bcl-xL induction. J. Immunol. 160:12.[Abstract/Free Full Text]
  22. Brunner, M. C., C. A. Chambers, F. K. Chan, J. Hanke, A. Winoto, J. P. Allison. 1999. CTLA-4-mediated inhibition of early events of T cell proliferation. J. Immunol. 162:5813.[Abstract/Free Full Text]
  23. Krummel, M. F., J. P. Allison. 1996. CTLA-4 engagement inhibits IL-2 accumulation and cell cycle progression upon activation of resting T cells. J. Exp. Med. 183:2533.[Abstract/Free Full Text]
  24. Greenwald, R. J., V. A. Boussiotis, R. B. Lorsbach, A. K. Abbas, A. H. Sharpe. 2001. CTLA-4 regulates induction of anergy in vivo. Immunity 14:145.[Medline]
  25. Gribben, J. G., G. J. Freeman, V. A. Boussiotis, P. Rennert, C. L. Jellis, E. Greenfield, M. Barber, V. A. Restivo, Jr, X. Ke, G. S. Gray, et al 1995. CTLA-4 mediates antigen-specific apoptosis of human T cells. Proc. Natl. Acad. Sci. USA 92:811.[Abstract/Free Full Text]
  26. Krummel, M. F., J. P. Allison. 1995. CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. J. Exp. Med. 182:459.[Abstract/Free Full Text]
  27. Egen, J. G., J. P. Allison. 2002. Cytotoxic T lymphocyte antigen-4 accumulation in the immunological synapse is regulated by TCR signal strength. Immunity 16:23.[Medline]
  28. Anderson, D. E., K. D. Bieganowska, A. Bar-Or, E. M. Oliveira, B. Carreno, M. Collins, D. A. Hafler. 2000. Paradoxical inhibition of T-cell function in response to CTLA-4 blockade: heterogeneity within the human T-cell population. Nat. Med. 6:211.[Medline]
  29. Oosterwegel, M. A., D. A. Mandelbrot, S. D. Boyd, R. B. Lorsbach, D. Y. Jarrett, A. K. Abbas, A. H. Sharpe. 1999. The role of CTLA-4 in regulating Th2 differentiation. J. Immunol. 163:2634.[Abstract/Free Full Text]
  30. Khattri, R., J. A. Auger, M. D. Griffin, A. H. Sharpe, J. A. Bluestone. 1999. Lymphoproliferative disorder in CTLA-4 knockout mice is characterized by CD28-regulated activation of Th2 responses. J. Immunol. 162:5784.[Abstract/Free Full Text]
  31. Saha, B., S. Chattopadhyay, R. Germond, D. M. Harlan, P. J. Perrin. 1998. CTLA-4 (CD152) modulates the Th subset response and alters the course of experimental Leishmania major infection. Eur. J. Immunol. 28:4213.[Medline]
  32. McCoy, K., M. Camberis, G. L. Gros. 1997. Protective immunity to nematode infection is induced by CTLA-4 blockade. J. Exp. Med. 186:183.[Abstract/Free Full Text]
  33. Borkow, G., Q. Leng, Z. Weisman, M. Stein, N. Galai, A. Kalinkovich, Z. Bentwich. 2000. Chronic immune activation associated with intestinal helminth infections results in impaired signal transduction and anergy. J. Clin. Invest. 106:1053.[Medline]
  34. Gusmao, R. D., A. M. Stanley, E. A. Ottesen. 1981. Brugia pahangi: immunologic evaluation of the differential susceptibility of filarial infection in inbred Lewis rats. Exp. Parasitol. 52:147.[Medline]
  35. Semnani, R. T., H. Sabzevari, R. Iyer, T. B. Nutman. 2001. Filarial antigens impair the function of human dendritic cells during differentiation. Infect. Immun. 69:5813.[Abstract/Free Full Text]
  36. Nutman, T. B., P. A. Zimmerman, J. Kubofcik, D. D. Kostyu. 1994. A universally applicable diagnostic approach to filarial and other infections. Parasitol. Today 10:239.
  37. Henry, N. L., M. Law, T. B. Nutman, A. D. Klion. 2001. Onchocerciasis in a nonendemic population: clinical and immunologic assessment before treatment and at the time of presumed cure. J. Infect. Dis. 183:512.[Medline]
  38. Kuhns, M. S., V. Epshteyn, R. A. Sobel, J. P. Allison. 2000. Cytotoxic T lymphocyte antigen-4 (CTLA-4) regulates the size, reactivity, and function of a primed pool of CD4+ T cells. Proc. Natl. Acad. Sci. USA 97:12711.[Abstract/Free Full Text]
  39. Tivol, E. A., F. Borriello, A. N. Schweitzer, W. P. Lynch, J. A. Bluestone, A. H. Sharpe. 1995. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3:541.[Medline]
  40. Takahashi, T., T. Tagami, S. Yamazaki, T. Uede, J. Shimizu, N. Sakaguchi, T. W. Mak, S. Sakaguchi. 2000. Immunologic self-tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J. Exp. Med. 192:303.[Abstract/Free Full Text]
  41. Steel, C., E. A. Ottesen. 2001. Evolution of immunologic responsiveness of persons living in an area of endemic bancroftian filariasis: a 17-year follow-up. J. Infect. Dis. 184:73.[Medline]
  42. Gopinath, R., L. E. Hanna, V. Kumaraswami, S. V. Pillai, V. Kavitha, V. Vijayasekaran, A. Rajasekharan, T. B. Nutman. 1999. Long-term persistence of cellular hyporesponsiveness to filarial antigens after clearance of microfilaremia. Am. J. Trop. Med. Hyg. 60:848.[Abstract]
  43. Klion, A. D., M. Massougbodji, B.-C. Sadeler, E. A. Ottesen, T. B. Nutman. 1991. Loiasis in endemic and non-endemic populations: immunologically mediated differences in clinical presentation. J. Infect. Dis. 163:1318.[Medline]
  44. Cooper, P. J., T. Mancero, M. Espinel, C. Sandoval, R. Lovato, R. H. Guderian, T. B. Nutman. 2001. Early human infection with Onchocerca volvulus is associated with an enhanced parasite-specific cellular immune response. J. Infect. Dis. 183:1662.[Medline]
  45. Steinbrink, K., E. Graulich, S. Kubsch, J. Knop, A. H. Enk. 2002. CD4+ and CD8+ anergic T cells induced by interleukin-10-treated human dendritic cells display antigen-specific suppressor activity. Blood 99:2468.[Abstract/Free Full Text]
  46. King, C. L., S. Mahanty, V. Kumaraswami, J. S. Abrams, J. Raganathan, K. Jayaraman, E. A. Ottesen, T. B. Nutman. 1993. Cytokine control of parasite-specific anergy in human lymphatic filariasis: preferential induction of a regulatory Th2 lymphocyte subset. J. Clin. Invest. 92:1667.
  47. Nutman, T. B., V. Kumaraswami. 2001. Regulation of the immune response in lymphatic filariasis: perspectives on acute and chronic infection with Wuchereria bancrofti in South India. Parasite Immunol. 23:389.[Medline]
  48. Mirshahidi, S., C. T. Huang, S. Sadegh-Nasseri. 2001. Anergy in peripheral memory CD4+ T cells induced by low avidity engagement of T cell receptor. J. Exp. Med. 194:719.[Abstract/Free Full Text]
  49. Kato, T., H. Nariuchi. 2000. Polarization of naive CD4+ T cells toward the Th1 subset by CTLA-4 costimulation. J. Immunol. 164:3554.[Abstract/Free Full Text]
  50. Alegre, M. L., H. Shiels, C. B. Thompson, T. F. Gajewski. 1998. Expression and function of CTLA-4 in Th1 and Th2 cells. J. Immunol. 161:3347.[Abstract/Free Full Text]
  51. Walunas, T. L., J. A. Bluestone. 1998. CTLA-4 regulates tolerance induction and T cell differentiation in vivo. J. Immunol. 160:3855.[Abstract/Free Full Text]
  52. Folkard, S. G., M. J. Taylor, G. A. Butcher, A. E. Bianco. 1997. Protective responses against skin-dwelling microfilariae of Onchocerca lienalis in severe combined immunodeficient mice. Infect. Immun. 65:2846.[Abstract]
  53. Herbert, D. R., J. J. Lee, N. A. Lee, T. J. Nolan, G. A. Schad, D. Abraham. 2000. Role of IL-5 in innate and adaptive immunity to larval Strongyloides stercoralis in mice. J. Immunol. 165:4544.[Abstract/Free Full Text]
  54. Martin, C., K. M. Al-Qaoud, M. N. Ungeheuer, K. Paehle, P. N. Vuong, O. Bain, B. Fleischer, A. Hoerauf. 2000. IL-5 is essential for vaccine-induced protection and for resolution of primary infection in murine filariasis. Med. Microbiol. Immunol. 189:67.[Medline]
  55. Taylor, M. J., R. P. van Es, K. Shay, S. G. Folkard, S. Townson, A. E. Bianco. 1994. Protective immunity against Onchocerca volvulus and O. lienalis infective larvae in mice. Trop. Med. Parasitol. 45:17.[Medline]
  56. Hogarth, P. J., S. G. Folkard, M. J. Taylor, A. E. Bianco. 1995. Accelerated clearance of Onchocerca microfilariae and resistance to reinfection in interleukin-4 gene knockout mice. Parasite Immunol. 17:653.[Medline]
  57. Bancroft, A. J., K. J. Else, R. K. Grencis, C. Martin, M. Saeftel, P. N. Vuong, S. Babayan, K. Fischer, O. Bain, A. Hoerauf. 1994. Low-level infection with Trichuris muris significantly affects the polarization of the CD4 response. Eur. J. Immunol. 24:3113.[Medline]
  58. Volkmann, L., M. Saeftel, O. Bain, K. Fischer, B. Fleischer, A. Hoerauf. 2001. Interleukin-4 is essential for the control of microfilariae in murine infection with the filaria Litomosoides sigmodontis. Infect. Immun. 69:2950.[Abstract/Free Full Text]
  59. Perez, V. L., L. Van Parijs, A. Biuckians, X. X. Zheng, T. B. Strom, A. K. Abbas. 1997. Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement. Immunity 6:411.[Medline]
  60. Steel, C., T. B. Nutman. 1993. Regulation of IL-5 in onchocerciasis: a critical role for IL-2. J. Immunol. 150:5511.[Abstract]
  61. Read, S., V. Malmstrom, F. Powrie. 2000. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J. Exp. Med. 192:295.[Abstract/Free Full Text]
  62. Levings, M. K., R. Sangregorio, M. G. Roncarolo. 2001. Human CD25+CD4+ T regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function. J. Exp. Med. 193:1295.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
R. T. Semnani, P. G. Venugopal, C. A. Leifer, S. Mostbock, H. Sabzevari, and T. B. Nutman
Inhibition of TLR3 and TLR4 function and expression in human dendritic cells by helminth parasites
Blood, August 15, 2008; 112(4): 1290 - 1298.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. D. Taylor, A. Harris, S. A. Babayan, O. Bain, A. Culshaw, J. E. Allen, and R. M. Maizels
CTLA-4 and CD4+CD25+ Regulatory T Cells Inhibit Protective Immunity to Filarial Parasites In Vivo
J. Immunol., October 1, 2007; 179(7): 4626 - 4634.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
B. Wan, H. Nie, A. Liu, G. Feng, D. He, R. Xu, Q. Zhang, C. Dong, and J. Z. Zhang
Aberrant Regulation of Synovial T Cell Activation by Soluble Costimulatory Molecules in Rheumatoid Arthritis
J. Immunol., December 15, 2006; 177(12): 8844 - 8850.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. Dent, I. Malhotra, P. Mungai, E. Muchiri, B. S. Crabb, J. W. Kazura, and C. L. King
Prenatal Malaria Immune Experience Affects Acquisition of Plasmodium falciparum Merozoite Surface Protein-1 Invasion Inhibitory Antibodies during Infancy
J. Immunol., November 15, 2006; 177(10): 7139 - 7145.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. D. Taylor, A. Harris, M. G. Nair, R. M. Maizels, and J. E. Allen
F4/80+ Alternatively Activated Macrophages Control CD4+ T Cell Hyporesponsiveness at Sites Peripheral to Filarial Infection.
J. Immunol., June 1, 2006; 176(11): 6918 - 6927.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Babu, C. P. Blauvelt, V. Kumaraswami, and T. B. Nutman
Regulatory networks induced by live parasites impair both Th1 and th2 pathways in patent lymphatic filariasis: implications for parasite persistence.
J. Immunol., March 1, 2006; 176(5): 3248 - 3256.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
V. Gillan and E. Devaney
Regulatory T Cells Modulate Th2 Responses Induced by Brugia pahangi Third-Stage Larvae
Infect. Immun., July 1, 2005; 73(7): 4034 - 4042.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
B. Sasisekhar, M. Aparna, D. J. Augustin, P. Kaliraj, S. K. Kar, T. B. Nutman, and R. B. Narayanan
Diminished Monocyte Function in Microfilaremic Patients with Lymphatic Filariasis and Its Relationship to Altered Lymphoproliferative Responses
Infect. Immun., June 1, 2005; 73(6): 3385 - 3393.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
S. Babu, V. Kumaraswami, and T. B. Nutman
Transcriptional Control of Impaired Th1 Responses in Patent Lymphatic Filariasis by T-Box Expressed in T Cells and Suppressor of Cytokine Signaling Genes
Infect. Immun., June 1, 2005; 73(6): 3394 - 3401.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. S. Satoguina, E. Weyand, J. Larbi, and A. Hoerauf
T Regulatory-1 Cells Induce IgG4 Production by B Cells: Role of IL-10
J. Immunol., April 15, 2005; 174(8): 4718 - 4726.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. D. Taylor, L. LeGoff, A. Harris, E. Malone, J. E. Allen, and R. M. Maizels
Removal of Regulatory T Cell Activity Reverses Hyporesponsiveness and Leads to Filarial Parasite Clearance In Vivo
J. Immunol., April 15, 2005; 174(8): 4924 - 4933.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
S. Specht, L. Volkmann, T. Wynn, and A. Hoerauf
Interleukin-10 (IL-10) Counterregulates IL-4-Dependent Effector Mechanisms in Murine Filariasis
Infect. Immun.,