Immunology 1992 77 277-283

Suppression by Trypanosoma cruzi of T-cell receptor expression by activated human lymphocytes M. B. SZTEIN & F. KIERSZENBAUM* Center for Vaccine Development, Department of Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland and *Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan, U.S.A. Acceptedfor publication 4 May 1992

SUMMARY The immunosuppression that develops during Chagas' disease and African sleeping sickness is thought to facilitate survival of the causative agents in their mammalian hosts. Whereas a number of manifestations of immunosuppression manifested during the course of these diseases has been reported in patients and animals, the mechanisms by which they are induced remain obscure. An in vitro system in which phytohaemagglutinin (PHA)-stimulated human peripheral blood mononuclear (PBMC) were co-cultured with purified Trypanosoma cruzi or T. brucei rhodesiense was used in the present work to establish whether these organisms were able to alter the capacity of activated helper/ inducer (CD4+) or cytotoxic/suppressor (CD8+) cells to express T-cell receptor (TcR). Suppressed interleukin-2 receptor (IL-2R), known to be caused by both the trypanosomes and supernatants containing their secretion products, was the independent parameter used to demonstrate the occurrence of immunosuppression in all experiments. We found marked reductions in the percentage of TcR+ cells in T. cruzi-containing cultures as early as 18 hr after PHA stimulation. This alteration was still readily demonstrable after 72 hr of culture, i.e. when last tested for. Suppressed TcR expression occurred concomitantly with reduced levels of CD4 or CD8 molecules on the surface of helper/inducer and cytotoxic/suppressor T lymphocytes, respectively, indicating that the parasite had induced more than one alteration in the same cells. These effects were reproduced when the trypanosomes were separated from the PBMC by a 0 45 ,m pore size filter or when filtrates from T. cruzi suspensions substituted for the parasite in the cultures, indicating that TcR suppression was mediated by a parasite secretion product(s). Interestingly, neither T. b. rhodesiense nor filtrates of suspensions of this organism altered significantly the level of TcR expression in cultures in which suppresed IL-2R expression by activated human T cells took place. Thus despite sharing the ability to impair IL-2R expression, T. cruzi and T. b. rhodesiense appear to differ in other mechanisms by which they affect human T-cell function. If occurring in infected hosts, the alterations that T. cruzi causes in the expression of TcR, CD4, CD8 and IL-2R-all molecules playing important roles in lymphocyte activation-could contribute to the development of the immunosuppression observed during the acute phase of Chagas' disease.

INTRODUCTION Chagas' disease and sleeping sickness result from two widely differing types of trypanosomal infections which have in common the development of immunosuppression during the acute stage in both patients and laboratory animals.'-9 Because immunological reactions can destroy Trypanosoma cruzi and

African trypanosomes, and the immune system helps contain the course of the infections caused by these organisms, diseaseassociated immunosuppression is believed to facilitate the multiplication and dissemination of these parasites in mammalian hosts. In recent years, a body of literature dealing with the specific immunological alterations that accompany infections by T. cruzi and African trypanosomes has accumulated. However, the mechanisms by which these anomalies are induced remain largely undefined. The use of an in vitro system in which stimulated lymphocytes are co-cultured with purified trypanosomes has made it possible to examine the types of cellular alterations induced directly by T. cruzi10'11 and T. brucei rhodesiense.'2" 3 Thus, human T lymphocytes stimulated with the T-cell-specific mitogen phytohaemagglutinin (PHA) manifest a

Abbreviations: FITC, fluorescein isothiocyanate; IL-2R, IL-2 receptor(s); PE, phycoerythrin; TIF, Trypanosoma cruzi immunosuppressive factor; TRIF, Trypanosoma brucei rhodesiense immunosuppressive factor. Correspondence: Dr M. B. Sztein, Center for Vaccine Development, Dept. of Pediatrics, University of Maryland, 10 S. Pine St, Baltimore, MD 21201, U.S.A.

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suppressed capacity to express interleukin-2 receptors (IL-2R) when co-cultured with either T. cruzi or T. b. rhodesiense trypomastigotes."" 2 Expression of IL-2R is essential for lymphocyte proliferation and the effective mounting of immunological responses.'4"'5 In addition, T. cruzi has been shown to affect the expression of CD4 and CD8, molecules which play key roles in the activation of helper/inducer and cytotoxic/suppressor T cells, respectively. 16 The T-cell receptor (TcR) is a heterodimeric molecule responsible for antigenic epitope recognition.7",8 Because alteration of TcR expression by infectious agents would have important implications in the immunological control of host defences against them, we studied whether T. cruzi, T. b. rhodesiense or their secretion products are able to tamper with expression of this cell surface component by activated human T-cell subpopulations.

MATERIALS AND METHODS Parasites Trypanosoma cruzi trypomastigotes (Tulahuen isolate) were purified from the blood of Crl-CD I (ICR) Swiss mice (Charles River Laboratories, Portage, MI) infected subcutaneously 9-11 days previously with 0-5-1 x 106 organisms. The flagellates were separated from blood cells by centrifugation (400 g, 20-, 45 min) over a mixture of Ficoll-Hypaque of specific gravity 1077 (Isolymph, Gallard-Schlesinger, Carle Place, NY)'9 followed by chromatography through diethylaminoethyl-cellulose.20 The recovered trypanosomes were washed twice with and resuspended in RPMI- 1640 medium (Gibco, Grand Island, NY) containing 100 IU of penicillin and 100 pg of streptomycin/ml, and supplemented with 5% heat-inactivated (56', 1 hr) foetal bovine serum (Sigma Chemical Co., St Louis, MO). This medium will be referred to as RPMI + 5 % FBS. The KETRI-2285 isolate of T. b. rhodesiense used in this work, originally from the Kenya Trypanosome Research Institute, was kindly provided by Dr A. B. Clarkson (New York University, NY). The organisms were initially stored under liquid nitrogen and then maintained by serial intraperitoneal passages in Crl-CD 1 (ICR) Swiss mice (Charles River Laboratories). The flagellates were purified from the blood of mice infected intraperitoneally 2 days previously with 0 5-1 x 106

organisms by chromatography through diethylaminoethylcellulose,2' using a buffer containing 103 mm Tris (pH 7-4), 1 5% glucose and 58 mm NaCI for column equilibration and elution. The recovered flagellates were washed twice by centrifugation with and resuspended in RPMI+ 10% FBS. Parasite concentrations were determined microscopically, using a haemacytometer. Trypanosoma cruzi and T. b. rhodesiense suspensions consisted of 100% trypomastigotes (> 99-5% viable, i.e. displaying motility).

Preparation of human peripheral blood mononuclear cells (PBMC) Purified PBMC from healthy volunteers were obtained by centrifugation of sterile heparinized blood through sterile Isolymph using the conditions described above. The viability of the PBMC used in our experiments was consistently >97%, determined by trypan blue exclusion.

Preparation of T. cruzi (TIF) and T. b. rhodesiense (TRIF) filtrates Suspensions containing 1 x I07 T. cruzi/ml in RPMI + 5 % FBS or 2 x 107 T. b. rhodesiense/ml in RPMI+ 10% FBS were incubated at 37° and 5% CO2 for 15-20 hr, and filtered through 0 45 pm pore size sterile filters. The T. cruzi filtrates, referred to as TIF, and those of T. b. rhodesiense, referred to as TRIF, were aliquoted and stored at - 20' until used.

Co-cultures of PBMC with T. cruzi or T. b. rhodesiense The volume of each culture was 1 ml. Cultures were set up in 24-well, flat-bottomed plates, using PBMC suspensions lacking or containing purified T. cruzi or T. b. rhodesiense, in the absence or presence of PHA (5 pg/ml; Sigma Chemical Co.). The final concentration of PBMC was 1 25 x 106 PBMC and that of parasites, where present, was 1 x 107 T. cruzi/ml or 2 x 107 T. b. rhodesiense/ml. The cultures were incubated at 370 and 5% CO2, and were harvested at various times (described under Results). The cells were washed by centrifugation using ice-cold phosphate-buffered saline solution, pH = 71 (PBS), and the cells were stained for flow cytometric analysis as described below. In experiments to test for the effect of TIF or TRIF on the expression of TcR, CD4 and CD8 by PHA-stimulated PBMC, 90% of the culture volume was replaced with either TIF or TRIF, all other conditions remaining unchanged. It is noteworthy that previous work has shown that the capacities of untreated and dialysed (versus fresh RPMI-1640 medium) TIF or TRIF preparations to suppress IL-2R expression are comparable.1'322 In some experiments, parasites were prevented from establishing physical contact with PBMC by using a Millicell HA filter (Millipore Corporation, Bedford, MA; pore size = 0 45 ,m) as described in detail previously.22

Immunofluorescence staining Cells were centrifuged and resuspended in ice-cold PBS containing 1 % bovine serum albumin and 0-1 % sodium azide for staining with monoclonal antibodies tagged with fluorescent dyes for two-colour flow cytometric analysis as described previously.'6'23 Briefly, aliquots of each cell suspension were incubated with a mixture of a fluorescein isothiocyanate (FITC)-labelled and a phycoerythrin (PE)-labelled monoclonal antibody in an ice bath for 30 min. The combinations of FITCand PE-labelled antibodies used in this work are described in the figure legends. Controls for background fluorescence were prepared with additional aliquots of each cell suspension subjected to similar treatments except for the use of FITClabelled and PE-labelled normal mouse IgG of the corresponding isotype. After two washings to remove unbound antibody, the cells were fixed with 1% formaldehyde and stored at 4° until analysed by flow cytometry. All experimental [FITC-anti-TcR 43, PE-anti-CD25 (anti-IL-2Rp55), PE-anti-CD4 and PE-antiCD8] and the appropriate isotypic control monoclonal antibodies were purchased from Becton Dickinson (San Jose, CA). Flow cytometric analyses Expression of surface antigens was determined by two-colour immunofluorescence using an EPICS ELITE flow cytometer/ cell sorter (Coulter Cytometry, Hialeah, FL) and the data were analysed using the Multi-2D software package (Phoenix Flow Systems, San Diego, CA). The excitation wavelength was 488 nm at 15 mW. Fluorescein isothiocyanate fluorescence was

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Effects of trypanosomes on T-cell receptor expression collected through a band pass filter of 510-540 nm and PE fluorescence through a band pass of filter of 560-590 nm. Erythrocytes, platelets, non-viable cells and parasites were excluded from analysis by setting an appropriate gate on the forward versus 900 light scatter parameters. Forty thousand cells were collected for each sample. Two-parameter data are displayed as density plots in a 4 x 4 decade logarithmic matrix. Areas within the dot density plots were integrated to determine the percentage of cells in the different quadrants. The mean channel number of the logarithm of the mean fluorescence intensity of the TcR+, IL-2R+, CD3+, CD4+ and CD8+ cell populations was used to compare the relative surface density of these molecules on lymphocytes incubated with the mitogens in the absence or presence of parasites or parasite supernatants. The percentage of variation in marker-positive cells in the presence of parasites was calculated by the equation: % variation = [(% positive cells in the presence of parasite material-% positive cells in the absence of parasite material)/(% positive cells in the absence of parasite material)] x 100, where the term parasite material represents living organisms, TIF or TRIF.

Lymphocyte proliferation assays Cultures of PBMC in 96-well plates (100 pl/well; 1 25 x 106 cells/ ml) were incubated at 370 and 5% CO2 in the presence or absence of PHA at 5 Mg/ml, together with or without T. cruzi or T. b. rhodesiense, for various periods of time, and pulsed with 1 pCi of tritiated thymidine ([3H]TdR; specific activity = 2 Ci/mmol; Amersham, Arlington Heights, IL)/well. The concentrations of parasites and the lengths of the [3H]TdR pulses are described in Results. In experiments designed to test the effect of TIF or TRIF on proliferation by PHA-stimulated PBMC, 90% of the volume of the culture was replaced by TIF or TRIF, all other conditions remaining unchanged. All cultures were terminated by automated harvesting and processed for liquid scintillation counting. Each condition was tested in quadruplicate. Presentation of results Each set of data presented in this paper is typically representative of at least two separate repeat experiments performed with cells from different donors.

RESULTS Suppression of TcR expression by T. cruzi As shown in Fig. 1, the presence in the culture of T. cruzi concentrations equal to or greater than 5 x 1 07 organisms/ml caused PHA-stimulated PBMC to express markedly lower levels of TcR than cells from parasite-free cultures. This suppressive effect was evidenced by marked reductions in both the percentage of TcR+ cells and the mean fluorescence intensity, a parameter which represents the cell surface density of the tested marker. The parasite concentrations significantly suppressing TcR expression were the same as those inhibiting IL-2R expression. Because titrations indicated that maximal suppression could be attained with levels > 1 x 107 T. cruzi/ml, concentrations ranging from 1 to 2 x 107 T. cruzi/ml were used in subsequent experiments, depending on parasite availability. It should be noted that a control determination of IL-2R expression was systematically included in our experiments to have

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independent evidence for the occurrence of suppression under the tested conditions. Although the results of this control may not be shown in subsequent figures to simplify data presentation, suppressed IL-2R expression was demonstrable in all instances and, in those cases where it was monitored, by reduced [3H]TdR incorporation as well. Kinetics of T. cruzi-induced suppression of TcR expression Studies were designed to define the earliest time of manifestation of T. cruzi-induced down-regulation of TcR expression. Significant reductions in the percentage of TcR+ cells were readily demonstrable at times . 18 hr after PHA stimulation; the degree of suppression increased with time up to 72 hr (i.e. the latest time at which it was tested) (Fig. 2). Of the three experiments in which we determinated TcR expression at earlier times, two showed suppression 4 or 6 hr after stimulation whereas the third failed to show any significant alteration (data not shown). Examination of the sets of data from most experiments revealed that reductions in the percentage of TcR+ cells occurring during the initial 24 hr were not accompanied by significant decreases in mean fluorescence intensity (a parameters representing cell surface density of TcR). However, at later times, both parameters were decreased. Concurrent suppression of TcR and CD4 or CD8 induced by T. cruzi To define the T-cell subpopulation(s) whose TcR-expressing capacity was affected by T. cruzi, we used two-colour flow cytometric analysis of TcR versus either CD4 or CD8 expression. As shown in Fig. 3, co-culture of PHA-stimulated PBMC with parasites resulted in a marked decrease in the proportion of cells expressing CD4 and TcR (CD4+ TCR+ cells). These changes were accompanied by declines in the surface density of TcR. Trypanosoma cruzi is known to downregulate CD4 expression by PHA-activated PBMC. 16 This observation was readily reproduced in the present experiments which also revealed marked reductions in the proportion of CD4+ cells and CD4 antigen density on the cells that remained CD4+. These changes were documented by rises in the proportions of CD4- TcR- cells and of dimmer CD4+ TcR+ cells.

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TcR - log fluorescence intensity Figure 2. Kinetics of suppression of TcR expression by T. cruzi. PHA-stimulated PBMC were incubated without or with 1 x 107 T. cruzi/ ml for the indicated amounts of time, harvested, stained for TcR and analysed by flow cytometry. The results are shown as single-colour histograms. The experiment showing data for 24,48 and 72 hr and that showing results for 18 and 38 hr were performed separately using

cells from different donors.

Similar changes and shifting patterns were recorded when the cells were stained for CD8 and TcR expression (Fig. 4). (a)

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Figure 3. Suppressed expression of TcR by PHA-stimulated CD4+ PBMC in the presence of T. cruzi. Cultures lacking (a) or containing (b) 2 x 107 T. cruzi/ml were incubated for 48 hr, harvested, stained and analysed by flow cytometry. Results are shown as posterior views of rotated two-colour isometric displays, with lateral projections.

Suppression of TcR expression by a T. cruzi secretion product(s) Because the suppressive effect of T. cruzi on IL-2R expression is mediated by a parasite-secreted protein,22 we asked whether suppressed TcR expression would be also mediated by a soluble factor(s). One of our approaches involved flow cytometric analysis of cells from cultures in which the organisms had been separated from the PBMC by using a Millicell-HA filter. As can be seen in Fig. 5A, suppressed TcR expression was demonstrable under these conditions. As in the case of parasite-PBMC co-cultures (Figs 3 and 4), organisms present in the filters suppressed TcR expression by either Thelper/inducer or Tcytooxic/ suppressor cells (data not shown). Preparations of TIF were also able to reproduce the suppressive effect of live T. cruzi (Fig. 5B).

Comparison of the effects of T. b. rhodesiense on TcR and IL-2R expression by PHA-stimulated PBMC We have recently reported that co-culture of mitogen-stimulated PBMC with T. b. rhodesiense suppresses IL-2R expression by PHA-stimulated PBMC as well as the ability of these cells to enter and progress through their cell cycle.'2'13 Therefore, it was of interest to find out if these organisms shared with T. cruzi the ability to suppress TcR expression. Neither the percentage of TcR+ PBMC nor the density of this receptor on the cell surface was affected significantly in any of five repeat experiments in which we used concentrations of T. b. rhodesiense which readily caused significant suppression of IL-2R expression (Fig. 6). Similar negative results were obtained by using up to 90% (vol./ vol.) TRIF in the cultures instead of live organisms under otherwise identical conditions (data not shown).

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Figure 6. Levels of expression of TcR (a) and IL-2R (b) by PHAstimulated PBMC alone (0) or co-cultured with 2 x 107 T. b. rhodesiense/ml (U) for 24 hr. Note that the level of IL-2R expression, but not that of TcR expression, was significantly reduced in the presence of T. b. rhodesiense.

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T. cruzi infection.24'25 The observation that both Thelper/inducer (CD4+) and Tcytotoxicisuppressor (CD8 +) lymphocytes had their TcRexpressing capacity impaired by T. cruzi points to the potential of this parasite to cause a major disarray in immune responsiveness.

Figure 4. Suppressed expression of TcR by PHA-stimulated CD8+ PBMC in the presence of T. cruzi. Cultures lacking (a) or containing (b) 2 x 107 T. cruzi/ml were incubated for 48 hr, harvested, stained and analysed by flow cytometry. Results are shown as posterior views of rotated two-colour isometric displays, with lateral projections.

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Figure 5. Suppression of PHA-induced PBMC proliferation by a secreted T. cruzi product(s). (a) 1-ml cultures lacking (PHA+[ ]) or containing (PHA + [Tc]) 2 x 107 T. cruzi separated from the PBMC by a Millicell-HA filter were incubated for 48 hr. (b) Cultures lacking or containing 90% TIF were incubated for 48 hr. After harvesting, the cells were stained for TcR and analysed by flow cytometry. Results are shown as single-colour histograms.

DISCUSSION These results uncover for the first time the ability of a microorganism to curtail the capacity of human T lymphocytes to express TcR molecules on their surface after activation. The relevance of this finding to Chagas' disease resides in the pivotal role that TcR plays in antigen recognition'7'8 and the importance of specific immune responses to host defence against

Two observations indicated that the expression of both CD4 and TcR or CD8 and TcR molecules were suppressed on the same cells. First, both the surface density of the tested molecules and the proportions of CD4+ TcR+ and CD8+ TcR+ cells were markedly suppressed by T. cruzi. Second, the proportions of CD4- TcR- and CD8-TcR- were concomitantly increased without rises in brightly stained, single-positive cell populations (i.e. CD4+ TcR- or CD8+ TcR-). TcR plays a crucial role in specific antigen recognition,' and there is an abundant literature showing the involvement of CD4 and CD8 molecules in a variety of lymphocyte activities.26-30 CD4 and CD8 participate in lymphocyte adhesion to antigen-presenting cells involving major histocompatibility complex class II and class I products, respectively, and in lymphocyte activation through interactions with the TcR complex.26 30 Furthermore, IL-2R expression is required for activated T cells to progress from GO/GI, to GIb phase of their cell cycle' and is, therefore, necessary for lymphocyte proliferation. When all of this information is considered together with the present results plus the fact that T. cruzi-induced suppression of IL-2R expression is accompanied by reductions in CD4 or CD8 levels on the same cells,'6 it becomes evident that the parasite can simultaneously affect activated lymphocytes in diverse and drastic manners. It is noteworthy that altered expression of post-activation molecules (i.e. IL-2R and transferrin receptors)"',' as well as the downregulation of surface proteins present prior to activation (i.e. CD3, CD4 and CD8) are demonstrable whether the PBMC are non-specifically activated with PHA or with monoclonal antiCD3, which mimics lymphocyte stimulation by specific antigen.32 Moreover, T. cruzi suppresses IL-2R expression by human lymphocytes activated with anti-CD2 monoclonal antibodies,33 indicating that this parasite affects lymphocyte activation regardless of the activation pathway. Any of these lymphocyte alterations, or combinations of them, if occurring in vivo, would suffice to interfere at several levels of the antigen recognition process, halting lymphocyte division' 34,35 and causing immunosuppression in T. cruzi-infected hosts. It is important to examine the data reported herein in the context of results presented previously. We have ruled out that the suppressive effects that T. cruzi induces on human lympho-

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cyte functions in our culture system are due to consumption of essential medium nutrients by the parasite.34 Furthermore, T. cruzi does not absorb, consume or destroy significant amounts of PHA and does not decrease the production of IL-2 by PHA-stimulated PBMC.34 In addition, T. cruzi does not affect IL-I secretion by monocytes and macrophages in either PHA-stimulated PBMC cultures or PBMC cultures stimulated with a bacterial lipopolysaccharide." It is also noteworthy that, over a 96-hr culture period, both the number and proportion of viable PBMC in T. cruzi-containing cultures are comparable with those present on parasite-free, control cultures whether or not PHA is present.1' Therefore, T. cruzi-induced suppression is not an artefact resulting from deficient medium conditions, insufficient IL-l or IL-2, removal of mitogen, or a reduction in the number of viable PBMC. That the suppressive effect of T. cruzi on TcR expression was also demonstrable when the organisms were prevented from establishing physical contact with the activated PBMC, or when cell-free filtrates of T. cruzi suspensions (TIF) substituted for the trypanosomes in our assay system, constituted clear evidence for the involvement of a soluble parasite secretion factor. These findings were in keeping with our previous observation showing suppression of IL-2R expression by PHA-stimulated PBMC under similar conditions." In that study, the secretion factor was found to be a non-dialysable molecule of protein nature, with a molecular weight between 30,000 and 100,000. It should be noted that undialysed TIF is as suppressive as TIF dialysed against fresh culture medium." Consequently, consumption of medium components by T. cruzi during TIF preparation is not the cause of its suppressive effects. Significant suppression of TcR expression was observed when using concentrations of T. cruzi as low as 5 x 106 parasites/ ml (Fig. 1), i.e. at a 4: 1 parasite: cell quotient. It is noteworthy that the presence of relatively large numbers of T. cruzi in lymphoid organs such as the spleen and lymph nodes of acutely infected patients and laboratory animals has been documented in numerous histopathology studies.36'37 Therefore, parasite: lymphocyte quotients equal to or greater than 4: 1 are likely to occur at infected foci in these organs. Both T. cruzi and T. b. rhodesiense markedly suppress IL-2R expression by activated human lymphocytes,"'2 thus preventing the latter cells from progressing through their cell cycle.'3 35 However, the contrasting behaviour of these two pathogenic trypanosomes with respect to their abilities to affect TcR expression under otherwise identical conditions points to a difference in the mechanisms by which they may manipulate the host's immune system. However, the experiments with T. b. rhodesiense were terminated within 24 hr after PHA stimulation due to the relatively rapid decline in parasite viability during culture. Therefore, we cannot rule out that, had larger numbers of T. b. rhodesiense remained alive for longer periods of time, a suppressive effect on TcR expression might have been noted. The alterations induced by T. cruzi are demonstrable within hours of lymphocyte activation. This might reflect the targeting by a parasite secretion product(s) of early processes such as, for example, Ca2+ fluxes, phosphatidyl inositol turnover, transcription of the genes coding for the suppressed surface molecules or messenger RNA stability. These possibilities are the subject of our current research. We would have liked to present information on whether T. cruzi or TIF also inhibit TcR expression by T cells activated

with a specific antigen. However, this was not possible because of the very small percentage of T cells specific for any antigen (usually < 2%) present in PBMC preparations, which contain a relatively large proportion (> 65%) of TcR+ cells. We were also precluded from doing studies with anti-CD3-stimulated PBMC cultures (which would have activated a larger proportion of T lymphocytes in an antigen-like fashion) because this treatment leads by itself to a reduction in the cell surface density of TcR which would have represented a major confounding factor. ACKNOWLEDGMENTS We thank Ms H. M. Lopez for her skilful assistance in the preparation and processing of biological materials used in some of our experiments and Mr M. K. Tanner for expert help with flow cytometric analyses. This work was supported by grant AI-26542 from the United States Public Health Service.

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