Gamma delta T cells in non-immune patients during primary schistosomal infection

The mevalonate pathway is critical for the survival of Schistosoma. γδ T cells, a small subset of peripheral blood (PB) T cells, recognize low molecular weight phosphorylated antigens in the mevalonate pathway, which drive their expansion to exert protective and immunoregulatory effects. To evaluate their role in schistosomiasis, we measured γδ T cells in the PB of non-immune travelers who contracted Schistosoma hematobium or Schistosoma mansoni in Africa. The maximal level of γδ T-cells following infection was 5.78 ± 2.19% of the total T cells, versus 3.72 ± 3.15% in 16 healthy controls [P = 0.09] with no difference between S. hematobium and S. mansoni in this regard. However, among the nine patients in the cohort who presented with acute schistosomiasis syndrome (AS), the level (3.5 ± 1.9%) was significantly lower than in those who did not (8.6 ± 6.4%, P < 0.05), both before and after therapy. Furthermore, γδ T cells increased significantly in response to praziquantel therapy. In a patient with marked expansion of γδ T cells, most expressed the Vδ2 gene segment, a hallmark of cells responding to cognate antigens in the mevalonate pathways of the parasite or the human host. These results suggest an immunoregulatory role of antigen responsive γδ T cells in the clinical manifestations of early schistosomal infection.


Introduction
Schistosomiasis is a helminthic infection caused by bloodflukes of the genus Schistosoma. The main species causing human disease are S. mansoni, and S. haematobium (in Africa) and S. japonicum (in Southeast Asia). Approximately 230 million people are infected with Schistosoma species in at least 76 countries, mainly in Africa [1]. The increasing number of travelers from industrialized to developing countries has resulted in acquisition of the disease by non-immune travelers from non-endemic areas as well. For example, in Israel, a country free of endogenous schistosomiasis, infection of young Israeli travelers to Africa (where the two dominant species are S. hematobium and S. mansoni), has resulted in a significant number of imported cases [2].
The initial type 1 helper T cell (T H 1)-response to the acute schistosomal infection targets adult parasites, but typically transitions to a T H 2-type response after the parasite's eggs are produced [3]. Perioval granulomatous inflammation ismediated by antigen specific CD4 þ T cells. The typically prevalent T H 2-type reaction is associated with mild lesions consisting of small granulomas comprised of eosinophils, macrophages, and lymphocytes with an increasingly fibrotic extracellular matrix. Tissue fibrosis, stimulated by interleukin (IL)-13 and other cytokines, can become pathological, a detrimental effect of chronic T H 2-type responses. The immune response to Schistosoma derived peptides is primarily mediated by activated CD4 þ T cell receptor (TCR) ab cells [4][5][6]. IL-4, 5, and 13 secreted by these cells, lead to eosinophilia, and help B cells to produce Schistosoma-specific antibodies, which are hallmarks of primary schistosomal infection [7].
Another subset of lymphocytes, gd T cells, are CD4-CD8-CD3þ T cells that express a TCR encoded by the g and d genes, and consist about $5% of the circulating PB T cells [8]. These cells are known to participate in the immune response against infectious organisms, and numerical perturbations of gd T cells exceeding 30% of the PB T cells in some cases, have been documented in a variety of infectious diseases [9]. In particular gd T cells that express a TCR encoded by the Variable (V) g9 and d2 genes recognize powerful microbial antigens, primarily (E)-4-hydroxy-3methyl-but-2-enyl pyrophosphate (HMBPP) produced in the alternative mevalonate pathway of some parasites and bacteria, and secrete primarily T1 type cytokines in response [10,11]. Isopentenyl pyrophosphate (IPP) is another albeit, less powerful, antigen produced in the mevalonate pathway of both eukaryotes and prokaryotes [11]. While gd T cells have been shown to be recruited to egg-induced granulomata during infection of experimental animals [12,13] evidence for involvement of gd T cells in human schistosomiasis is very limited. The one publication broaching this subject reported that gd T-cells are expanded in the PB of patients with bladder cancer related to chronic schistosomiasis relative to bladder cancer patients without schistosomiasis [14].
Because the mevalonate pathway appears to play a critical role in the survival of Schistosoma, and has even been proposed as a therapeutic target, the gd T cell response to human schistosomiasis is of great potential interest [15]. Thus, the goal of this study was to evaluate gd T-cells in PB of previously healthy non-immune individuals with primary schistosomal infection, and to correlate the level of these cells in the PB with the clinical syndromes developing in the patients.

Patients
The 18 patients included in the study were non-immune travelers who contracted infections with either S. mansoni or S. hematobium for the first time during their travel in Africa in an area endemic for schistosomiasis. All patients were seen in the Center for Geographical Medicine at the Sheba Medical Center upon returning from their trip to endemic areas after developing symptoms of AS. The asymptomatic patients were companions to the same exposure of the symptomatic patients who came for serology screening to assess whether they too were infected.
Diagnosis was made by detecting eggs in the urine or stool, and/or by serology performed at the division of parasitic diseases at the Center for Disease Control (Atlanta, USA). All sera were initially screened by the FAST-enzyme linked immunosorbent assay (ELISA), positive sera were considered those registering >8 units and they were confirmed by immunotransfer blot. The serology test is highly sensitive (99%) and specific (99%) [16,17]. All patients in the study tested positively for antibodies by this methodology.
Treatment consisted of a course of praziquantel (60 mg/kg in two divided doses). Controls were healthy people matched for age and sex. Blood samples were drawn for evaluations of complete blood count (CBC), schistosomal serology, and T-cell antigens upon the first clinic visit, and for 10 patients, 2-4 weeks after treatment (one patient had evaluation only after treatment). Immunological study of gd T cells was approved by the Helsinki committee of the Sheba Medical Center. The other tests of individual patients was carried out as and when they presented and independently of the others.

Determination of lympocyte subsets
Peripheral blood mononuclear cells (PBMC) were stained with monoclonal antibodies (mAb) TCR1 [directed against constant region of TCRg chain (Cg)], that identifies all gd T cells, or with mAb to Vd1 or Vd2 in individual instances ( Fig. 3), to identify mutually exclusive gd T cell subsets expressing the Vd1 or Vd2 gene segments. Percent of total gd cells, or of the Vd1 or Vd2 subsets within the total T cell population within the gated lymphocyte (L) population (above background stain with isotype control mAb) was recorded using the installed computer software. To obtain percent of gd T cells of all CD3þ T cells, the percent of those staining with mAb to gd TCR or V regions within the gated population, was divided by percent of CD3þ T cells within the same gate. All mAb were purchased from T cell Sciences (Cambridge, Massachusetts, USA), and analyzed on an Epics profile II Coulter Electronic FACS as described previously [18]. MAb to CD4, CD8, and CD20 were from Becton Dickinson. A single analysis was performed for each patient. Differential CBC for enumeration of eosinophils were performed on an automated Coulter Counter.

Statistics
Values in compared groups were normally distributed (Shapiro-Wilk test). One and two tailed Student's T-test to compare mean values and Pearson's correlation coefficient between groups of values were computed using Excel software. Means (M) and 1 SD were calculated and reported as M AE 1 SD.

Patient and controls
Fifteen male and three consenting female patients, (mean age 26.8 AE 8.9 years), six with S. mansoni, 11 with S. hematobium, and one with mixed infection (Table 1) were studied. None had previously been exposed to Schistosoma.
All patients had evidence of a humoral immune response to schistosomal antigens at diagnosis, reflected by the presence of antibodies in the serum detected by ELISA and immunoblot. Eggs were recovered in urine or stool of six patients. Thirteen of the 18 patients had symptoms attributable to the disease (acute or chronic manifestations), whereas five were asymptomatic (Table 1).

gd T cell response
The mean of the highest percentage of gd T cells measured either before or after treatment among the 18 patients was 5.7 AE 2.1% of the PB CD3þ T cells compared to a control cohort of 16 healthy individuals (mean 3.7 AE 3.1%, P ¼ 0.09) suggesting a trend toward an elevated level of gd T cells in these patients during infection. There was no significant difference between the mean of the maximal percentage of gd T cells among the total peripheral blood CD3þ T cells among S. mansoni and S. hematobium infected patients (4.1 AE 3.4% vs. 3.7 AE 1.8%, respectively) or in the mean for all evaluations performed in the respective patient groups (4.6 AE 3.5% vs. 6.2 AE 6.3%, P ¼ ns, Fig. 1a). Patients with acute schistosomiasis (ASþ) presented nonsignificantly earlier than those without (ASÀ) (3.2 AE 2.0 vs. 8.8 AE 10.8 months, P ¼ 0.14), but there was no correlation between time of presentation when gd T cells were evaluated, and maximal levels of gd T cells in either ASþ or ASÀ patients (R ¼ 0.49, P ¼ not significant, R ¼ À0.09, P ¼ not significant, respectively).
Interestingly however, percent of gd T cells among PB T cells of the nine ASþ patients (n ¼ 15 evaluations) was significantly lower than in the nine ASÀ patients (n ¼ 15 evaluations) (3.0 AE 1.7% vs. 7.4 AE 5.3%, respectively, P < 0.005) (Fig. 1b). Furthermore, comparison of the means of the highest percentages of gd T cells among CD3þ T cells in the PB, measured in each of ASþ patients when one or more evaluations was performed, was also lower than in the ASÀ patients (3.2 AE 1.9% vs. 8.0 AE 6.2%, P < 0.045). Likewise, for evaluations done either before or after therapy, the percent gd T cells was higher in the ASþ group (Fig. 2). In contrast to the significant differences in percent gd T cells among the CD3þ T cells, and although the mean in ASþ patients was higher, the absolute number of eosinophils in the complete white blood cell counts of the two groups was not significantly different (2148 AE 1516 vs. 1278 AE 1345 P ¼ 0.2) (Fig. 1c). In addition we found that, in both ASÀ and ASþ patients, gd T cells expanded significantly after therapy (Fig. 2). Finally, to determine whether cells expanding in the patients bear characteristics consistent with a response to antigens produced in the mevalonate pathway, we stained the PBMC of two ASÀ patients, the first with elevated PB gd TCR expressing cells (11.8% of lympocytes) and the second with no expansion of gd T cells (2.2%) using mAb to the Vd2 and Vd1 gene products expressed in gd TCR. As shown in Figure 3, in the patient with expanded gd T cells, >90% expressed Vd2 and only <10% expressed the Vd1 gene segment. In contrast, among the T cells in the patient with no relative gd T cell expansion there was a more even distribution of Vd2þ and Vd1þ lymphocytes. As shown in Figure 3, these patients had similar percentages of B cells, (CD20þ), and of CD8þ and CD4þ T cells in the peripheral blood lymphocyte population. We also examined gd T cells in relation to the development of pulmonary infiltrates (PI) in the patient cohort. Percent of gd T cells among all peripheral blood T cells in patients with PI were lower than in patients without PI, although the differences were not statistically significant.

Discussion
This is the first reported study, to our knowledge, of gd T cells in non-immune patients with schistosomiasis, revealing differential responses in patients in accordance with the development or lack of the clinical syndrome of acute schistosomiasis. This syndrome occurs in a subset of non-immune patients exposed to Schistosoma-infected water and is also known as Katayama syndrome. Clinical manifestations include fever, cough, accompanied by pulmonary infiltrates, fatigue, arthralgias and myalgias, urticarial rash, angioedema, and abdominal pain [19]. The syndrome is unique to the non-immune population (usually travelers),   appears 3-8 weeks after exposure to the Schistosoma cercaria, and may be related to immune complexes containing schistosomal antigens. In healthy individuals, the percent of gd T cells within the total CD3þ T cell population is generally stable over time, and only change in specific stimulatory circumstances, for example, when patients are treated with intravenous administration of bisphosphonate for osteoporosis, which increases monocyte expression of IPP, a gd T cell antigen, or in multiple sclerosis patients with active brain disease, by unknown mechanisms [20,21]. Our results now reveal for the first time, a relative expansion of gd T cells within the total CD3þ T cell population in the PB in a group of patients from non-endemic areas who were infected with Schistosoma, following treatment of the infection. The drive for this expansion is currently unknown, but could be due to Schistosoma derived antigens. In this regard, our data, based on a patient in whom there was a large relative expansion of these cells, revealed that the majority of the relatively expanding gd T cells expressed the Vd2 gene.  [22]. Thus, strong gd T cell antigens such as IPP and its isomer, dimethylallyl pyrophosphate (DMAPP), are indeed produced by this parasite and could be responsible for gd T cell activation and expansion within the peripheral blood T cell pool [23]. The differential increased relative expansion of gd T cells in the PB in patients without, relative to patients with, the AS clinical syndrome even before therapy (Fig. 2) could be explained by host and parasite factors. For example, parasite loads and their metabolic activity, may differ between patients. For example, in acute Mansoni schistosomiasis studied in 26 Puerto Rican patients, severity of illness was found to be positively correlated (r ¼ 0.79) with the intensity of infection as measured by the concentration of eggs of S. mansoni in stool specimens [24]. Compounded with our results indicating that in these patients gd T cells would be less likely to expand, this suggests that in patients who develop the acute syndrome the ability of gd T cells to proliferate in response to their cognate antigens is suppressed, perhaps due to the strong Th2 TCRab mediated response to the increased parasitic load. A similar low level of gd T cells in patients exhibiting a potent Th2 like response is likewise found in allergic individuals, who have decreased gd T cells in their PB [25]. It is possible, however, that in individuals manifesting clinically with AS, gd T cells are distributed in the tissues where parasites localize, similar to their redistribution to the airways in allergic asthmatics [26].
On the other hand, the absence of systemic symptoms associated with a higher relative level of gd T cells among the T cells in the peripheral blood, suggests that these cells may function to dampen the systemic acute inflammatory response engendered by the parasite, which is reminiscent of previous finding showing inverse relationship of gd T cells and intensity of inflammation in juvenile idiopathic arthritis [27,28]. Recent studies suggest that gd T cells could indeed suppress ab T cells [29]. The observed increase of gd T cells after therapy was instituted (Fig. 2), when the clinical syndrome is subsiding, together with data indicating that gd T cells increase in the PB of malaria patients after therapy, lend additional support to this concept [18].
Further studies of the involvement of gd T cells in schistosomal infections could lead to novel understanding of the host-parasite interactions and new therapeutic modalities. For example, bisphosphonates can upregulate IPP by blocking isopentenyl pyrophosphate synthase, thus boosting the gd T cell response, which may have a beneficial clinical effect in patients with AS [15].