T-Cell-Independent and T-Cell-Dependent B-Cell Responses to

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1 INFECTION AND IMMUNITY, JUlY 1990, p Vol. 58, No /90/ $02.00/0 Copyright C 1990, American Society for Microbiology T-Cell-Independent and T-Cell-Dependent B-Cell Responses to Exposed Variant Surface Glycoprotein Epitopes in Trypanosome-Infected Mice DAVID M. REINITZ AND JOHN M. MANSFIELD* Department of Veterinary Science, University of Wisconsin-Madison, 1655 Linden Drive, Madison, Wisconsin Received 12 December 1989/Accepted 10 April 1990 The T-cell dependency of B-cell responses to variant surface glycoprotein (VSG) epitopes exposed in their native surface conformation on Trypanosoma brucei rhodesiense clone LouTat 1 was investigated. T-cell requirements were examined by analyses of gamma globulin preparations derived from trypanosome-infected BALB/c nude (nulnu) and thymus-intact (nu/+) mice. A radioimmunoassay was used to selectively quantitate antibody binding to native VSG 1 epitopes present on the surface of viable trypanosomes. Such analyses of VSG-specific antibody in infected mice demonstrated that in the absence of T cells there was a significant B-cell response to exposed VSG epitopes; however, in the presence of T cells these surface epitope-specific responses were greatly enhanced. In contrast to infection, immunization of mice with purified VSG 1 or paraformaldehyde-fixed parasites elicited significant VSG surface epitope-specific responses only in the presence of T cells (i.e., in nul+ mice only). VSG-specific antibody responses in mice infected with three other clonal T. brucei rhodesiense populations (LouTat 1.2, 1.5, and 1.9) were found to be similar in this pattern, although not identical, to the anti-loutat 1 responses. An important exception was that mice infected with LouTat 1.8 required T cells to produce VSG surface-specific antibody. Thus, the VSG surface epitope-specific B-cell responses in trypanosome-infected mice represent composite T-cell-independent and T-cell-dependent processes, and a significantly stronger response is made in the presence of T cells. However, immunization with VSG in the absence of infection elicited only T-cell-dependent responses. Since the relative contribution of T-cell-independent and T-cell-dependent processes to the total VSG-specific antibody produced during infection was variable (as seen with the absence of a T-cell-independent response to LouTat 1.8), this may reflect differences in the primary structure or display of VSG molecules on the trypanosome membrane or may represent active parasite interference with some epitope-specific B-cell responses. African sleeping sickness is caused by infection with the protozoan parasites Trypanosoma brucei rhodesiense and T. brucei gambiense. Approximately 50 million Africans living in sub-saharan regions are at risk of being infected (7). The blood-borne trypanosomes evade temporally protective immune responses by transcriptional switching of genes encoding the variant surface glycoprotein (VSG), which covers the plasma membrane of the parasite (5). Studies of VSG gene expression have revealed that the regulation of these genes is among the most unusual and complex of eucaryotic systems studied to date (8). The interaction of the parasite with cells and products of the immune system also is extremely complex. To better understand basic immunological characteristics of this infectious disease, it is one of the long-term goals of this laboratory to precisely map B- and T-cell epitopes on the VSG molecule that are involved in temporally protective immune responses. Previous studies from this laboratory (19) demonstrated that independent regulation of B-cell responses to surface and subsurface epitopes on the VSG molecule occurs during infection. However, the T-cell requirements of these B-cell responses are poorly understood. Rigorous determination of T-cell involvement in these responses should clearly precede detailed attempts to map potential T-cell epitopes. Earlier studies have shown that athymic nude mice can control T. brucei rhodesiense (1, 9, 14), T. brucei brucei (3), and T. congolense (17) infections but not infections with unrelated * Corresponding author trypanosomes such as T. musculi (18). Such findings demonstrated that thymus-independent (TI) B-cell responses artificially isolated from thymus-dependent (TD) mechanisms are sufficient to control relapsing parasitemias; however, their contribution to the overall B-cell response in the normal thymus-intact host is unclear. A quantitative comparison of VSG-specific B-cell responses in infected athymic (nulnu) and thymus-intact (nul+) mice may directly determine the relative contributions of TI and TD processes to the temporally protective immune response. The present study addresses two basic questions concerning the regulation of VSG surface epitope-specific B-cell responses. First, what are the T-cell requirements for B-cell responses mounted to VSG epitopes exposed in their native conformation on viable trypanosomes during infection, and are they similar after immunization with purified VSG preparations? Knowledge of such basic characteristics of these temporally protective immune responses is of interest in its own right and is relevant in the context of future investigations (above). Second, are the patterns of TI or TD VSGspecific B-cell responses observed in clonal trypanosome populations immutable? That is, are the general patterns of such responses the same for each variant population to arise during infection, or are there marked differences that may be attributable to differences in VSG structure or in parasite immunodulatory effects? Such a broader picture of TD B-cell function is relevant to the disease process, because the host must mount successive VSG-specific B-cell responses to many variant populations.

2 2338 REINITZ AND MANSFIELD MATERIALS AND METHODS Mice. Athymic nude (nulnu) BALB/c mice and their thymus-intact (nul+) littermates were obtained from the University of Wisconsin Gnotobiotic Laboratory. Mice were age matched (8 to 10 weeks) and weighed 20 to 25 g at the onset of all experiments. Outbred mice, used for expanding cryopreserved trypanosome clonal populations (stabilates), were obtained from the University of Wisconsin Charmany Farms mouse colony. All mice were housed in American Association for Accreditation of Laboratory Animal Careapproved facilities and were handled according to National Institutes of Health guidelines. Infection with trypanosomes. Frozen stabilates of T. brucei rhodesiense clones LouTat 1, 1.2, 1.5, 1.8, and 1.9 were thawed and expanded in cyclophosphamide (Cytoxan, 300 mg/kg of body weight; Mead Johnson and Co., Evansville, Ind.)-immunosuppressed outbred mice. When these mice exhibited systemic parasitemias of 2 x 108 to 5 x 108 trypanosomes per ml (typically in 3 to 5 days), they were exsanguinated, and parasites were purified from heparinized blood by DEAE-cellulose chromatography (10). Infections were subsequently initiated in nude and thymus-intact mice by intraperitoneal injection of 106 washed viable trypanosomes in 300,u of PBS (10 mm P04 [ph 8.0], 150 mm NaCl)-1% glucose. Immunization of mice. Paraformaldehyde-fixed trypanosomes used for immunization were prepared by incubation of purified live parasites in 1% paraformaldehyde-150 mm NaCl (ph 7.4) for 1 h at 20 C, followed by washing four times in PBS (10 mm P04, 150 mm NaCl) at ph 7.4. BALB/c nulnu and nul+ mice were injected intraperitoneally with up to 1010 fixed trypanosomes. In addition, BALB/c mice were immunized with up to 500 jig of VSG isolated from clone LouTat 1, 1.2, 1.5, 1.8, or 1.9 as previously described (20). Briefly, mice were injected intraperitoneally with 500,ug of purified VSG (see below) in PBS and exsanguinated 10 days later (see Results). VSG used for immunization was purified from trypanosome clones after distilled water lysis in the presence of ZnCl2 and phenylmethylsulfonyl fluoride (4). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single Coomassie blue-stained band (Mr, 61,000 to 65,000, depending upon the variant population) when 30,ug of protein was loaded per lane. The purified proteins in approximately 3 ml were dialyzed against two changes of PBS (ph 7.4) (2 liters per 24 h), and their A280 was determined by using the PBS on the outside of the dialysis bag as a spectrophotometer blank. Exactly 3,000 RI of protein solution (1 mg/ml) and the PBS blanks were placed in separate preweighed vessels, lyophilized, and weighed; the difference was assumed to be the weight of the VSG. Extinction coefficients were then calculated as E2nm (i.e., the A280 of a 10-mg/ml solution) for the LouTat 1 (E = 6.71) and 1.5 (E = 4.67) VSG molecules. These values were used to determine the quantity of VSG for immunization of BALB/c nude mice. Titration of anti-y and anti-,u reagents. Since a comparison of nulnu and nul+ immune responses was the basis of this study, it was imperative to ensure that the assay procedure used was not significantly biased toward detection of VSGspecific immunoglobulin M (IgM) (i.e., nulnu responses) versus immunoglobulin G (IgG) antibody binding to the surface of any LouTat clonal parasite populations. To address this, the affinity-purified 125I-labeled rabbit anti-mouse reagent was a carefully titrated mixture of anti-,u and anti--y INFECT. IMMUN. preparations. This 125I-labeled reagent was then tested in control solid-phase assays with affinity-purified IgG and IgM antibodies. The rabbit anti-y reagent was made in our laboratory by using a mouse normal gamma globulin agarose affinity column (Affi-Gel 10) and accordingly detected primarily IgG; however, it did possess to a lesser extent some anti-,u activity. To allow equivalent detection of,u and -y isotypes, this reagent was spiked with commercially available affinity-purified rabbit anti-mouse IgM (no ; Cooper Biomedical). Various ratios of these two affinitypurified reagents (1:100 to 100:1) were iodinated and used in solid-phase assays. Dynatech Immulon II wells were coated separately with affinity-purified mouse IgG or IgM (no or M1520, respectively; Sigma Chemical Co., St. Louis, Mo.) (50 jig/ml in PBS) and washed three times; 5 x 104 cpm of the rabbit anti-mouse reagent (,uand -y at various ratios) in PBS with 0.1% bovine serum albumin was added, and preparations were allowed to incubate for 2 h at 4 C. The wells were then washed three times in PBS with 0.1% bovine serum albumin, and the bound radioactivity was determined by using a gamma counter. When the anti--y globulin and anti-,upreparations were iodinated and used at a ratio of 40:1 (vol/vol), the mixture would bind 18, cpm in wells coated with either IgG or IgM. Furthermore, in wells coated with 25 p,g of each isotype per ml, the titrated reagent bound 32, cpm. All subsequent iodinations in the study used a 40:1 (vol/vol) mixture of these anti--y globulin and anti-,u reagents. Thus, wells coated with 50,ug of IgG or IgM bound equal amounts of the titrated 1251I-labeled reagent. Furthermore, Immulon II wells are coated to nonspecifically bind all proteins equivalently. These data indicate that IgG and IgM bind similarly if not equally; however, the actual amounts bound are unknown. Quantitation of surface-specific B-cell responses by RIA. A radioimmunoassay assay (RIA) was developed in this laboratory (19) that allowed quantitation of antibody binding to exposed VSG epitopes presented on the surface of live trypanosomes. Briefly, fresh whole serum samples from trypanosome-infected or immunized BALB/c mice were clarified of lipoproteins by dextran sulfate treatment, and the gamma globulin fractions were isolated by ammonium sulfate precipitation followed by dialysis against PBS (20). These purified gamma globulin fractions exhibited much lower background binding than whole sera when live trypanosomes were used as binding targets. On the day of assay, fresh parasites of the relevant variant type were isolated from the blood of immunosuppressed mice (10), suspended in PBS-1% glucose with 20% heat-inactivated fetal bovine serum, and aliquoted into 96-well vinyl assay plates (no. 2595; Costar, Cambridge, Mass.) at 5 x 107 parasites in 50,u per well. Then 50-,ul samples of various dilutions of the purified gamma globulin preparations added and incubated were at 4 C for 1 h with gentle shaking. The plates were then washed two times (10 min, 1,500 x g), suspended in 50,ul of PBS-1% glucose-20% heat-inactivated fetal bovine serum (per well) with 105 cpm of 1251I-labeled rabbit anti-mouse gamma globulin (specific activity, 21 X 109 cpm/mg; -y and,u at 40:1), incubated as above, and washed three times. Plates were subsequently cut apart into individual wells, and bound radioactivity was determined by using a gamma counter. The affinity-purified rabbit antimouse antibody was iodinated with lodogen (Pierce Chemical Co., Rockford, Ill.) as previously described (20). Control wells in each assay included normal gamma globulin and immune mouse variant-specific antibody preparations. All

3 VOL. 58, 1990 TI AND TD VSG-SPECIFIC B-CELL RESPONSES 2339 values represent the means of triplicate or quadruplicate values. RESULTS Dose responses to paraformaldehyde-fixed trypanosomes and purified VSG. Before comparative studies of nulnu or nul+ mice immunized with purified VSG or paraformaldehyde-fixed parasites, various control experiments were conducted to validate the assay procedures. Initially, a doseresponse experiment was done to determine the optimal amounts of the immunogens used for maximal stimulation and to ensure that the same dose was equally effective in nulnu and nul+ animals. Three mice (nulnu or nul+) were immunized with 10, 100, or 500,ug of purified VSG or with 106, 108, or 1010 paraformaldehyde-fixed trypanosomes, and gamma globulin was prepared 10 days later. Day 10 was chosen based on previous kinetic analyses of surface-specific anti-vsg responses in B1O.BR and C3HeB/FeJ mice (19; see below). These preparations were assayed at a 1:10 dilution (see Materials and Methods) for binding to native surface-exposed VSG epitopes displayed on live LouTat parasites. Immunization with 1010 fixed parasites or with 500,ug of purified LouTat 1 VSG consistently elicited maximal activity against surface-specific VSG. Typical results after immunization of nu/+ mice with 10, 100, or 500 plg of purified LouTat 1 VSG were 1, , 1, , and 3, cpm bound, respectively. In addition, the binding activity of gamma globulin (diluted 1:10) obtained after immunization with 106, 108, or 1010 fixed parasites was , , and 3,410 ± 330 cpm, with background counts of approximately 1,000 cpm (Fig. 1 and 2). Thus, 500 jig of purified LouTat 1 VSG and a dose of 1010 fixed trypanosomes were used to generate the data in Fig. 1. The nulnu mice did not produce detectable anti-vsg activity on day 10 after immunization with any of these doses (Fig. 1). To examine further, mu globulin (prepared as above) was harvested after 7 and 14 days and assayed as above; however, as on day 10 no anti-vsg activity was detectable at these times after immunization. Kinetics of antibody production in infected nu/nu and nu/+ mice. Before comparative studies of the immune responses to various LouTat parasite clones (1, 1.2, 1.5, 1.8, and 1.9), a brief examination of the kinetics of these responses in nulnu versus nul+ mice was conducted. Three mice (nulnu or nul+) were infected with 106 live LouTat 1 parasites and gamma globulin was prepared 7, 10, or 14 days later and assayed for antibody binding to VSG epitopes exposed on live trypanosomes, as previously described (19) (see Materials and Methods). Binding data revealed similar but not identical kinetics of antibody production in nulnu and nul+ animals. Although both substrains produced maximal specific antibody concentrations on day 10 and lower levels on day 14, the nulnu mice produced slightly more VSG surface epitope-specific antibody on day 7 than did nul+ animals. Typical results from the binding assay for nulnu and nul+ gamma globulin samples were as follows: day 7, 2,820 ± 475 and 2,355 ± 475 cpm; day 10, 3,210 ± 390 and 3,370 ± 440 cpm; and day 14, 2,130 ± 475 and 2,270 ± 275 cpm. These binding values and kinetics were similar to those previously measured in B1O.BR and C3HeB/FeJ mice infected with T. rhodesiense (LouTat 1 or 1.5) and C57BL/6 nulnu mice infected with T. congolense (17, 19). Since both the nulnu and nul+ mice produced peak serum concentrations of VSG surface epitope-specific antibody 10 days postinfection, this time point was chosen for all comparative studies (Fig. 1 and o 3.0- z 0 CL Log [Sera Dilution] FIG. 1. Quantitation of TI and TD B-cell responses specific for native VSG surface-exposed epitopes. BALB/c athymic (nulnu) or thymus-intact (nul+) littermate mice were infected or immunized (three mice per treatment) with T. brucei rhodesiense clone LouTat 1, and gamma globulin fractions were derived from day 10 pooled serum samples. Dilutions of these preparations were incubated with freshly isolated viable trypanosomes, which were subsequently washed and incubated with an affinity-purified '25I-labeled rabbit anti-mouse immunoglobulin reagent (,u and -y specific). The parasites were then washed, and bound radioactivity was determined with a gamma counter (see Materials and Methods). (A) Background binding by normal gamma globulin before infection; (B) aggregate TI and TD binding activity of day 10 gamma globulin derived from BALB/c nulnu (TI) or nul+ (TI and TD) mice infected with 106 LouTat 1 trypanosomes; (C) exclusive TD binding displayed by gamma globulin derived from mice immunized with 500 p.g of purified VSG; (D) exclusive TD binding by gamma globulin derived from mice immunized with 1010 paraformaldehyde-fixed LouTat 1 parasites. 2). In addition, at day 10, both the nulnu and nul+ animals have cleared the first systemic parasitemia and the number of trypanosomes per milliliter of blood is relatively low (<105 cells per ml) (5, 6, 9). Thus day 10 postinfection represented a valid time point to harvest VSG surface epitope-specific antibody; both nu/nu and nu/+ mice produced peak concentrations in serum, and parasitemias were at an equivalent and low level. Nude mouse B-cell responses to trypanosome VSG. BALB/c nude (nulnu) mice or their thymus-intact (nul+) littermates were infected with 106 live parasites or were immunized with purified VSG (500,ug) or paraformaldehyde-fixed trypanosomes (1010 cells). Ten days after immunization or infection, sera were collected and then analyzed for the presence of antibody binding to native exposed VSG epitopes displayed on the surface of the live parasites. The kinetics of the BALB/c nul+ response after immunization or infection was examined, and day 10 was found to represent the maximal concentrations in serum, whereas the nulnu mice did not respond to immunization (see above). An RIA employing freshly isolated live trypanosomes as binding targets, which selectively detects only surface specific B-cell responses (and not buried VSG epitope specific responses), was used to assay gamma globulin preparations derived from the nude and thymus-intact mice (19). Figure 1 shows the RIA results

4 2340 REINITZ AND MANSFIELD z 7.0 :L 5.0 D Log [Sera Dilution] FIG. 2. Differential VSG surface epitope-specific B-cell responses elicited during active infection with clonal populations of LouTat trypanosomes. Groups of BALB/c athymic (nulnu) or thymus-intact (nul+) littermate mice (three mice per treatment) were infected (106 parasites) with one of four clonal populations of LouTat parasites (1.2, 1.5, 1.8, 1.9), which had been isolated previously at various times (10 to 50 days) from mice with relapsing parasitemias after a LouTat 1-initiated infection. The gamma globulin preparations derived from the nulnu and nul+ mice infected with LouTat 1.2, 1.5, or 1.9 trypanosomes all displayed significant binding to the respective VSG surface-exposed epitopes. However, infection with LouTat 1.8 elicited specific binding activity exclusively in nul+ animals. Thus, aggregate B-cell responses to LouTat 1.2, 1.5, and 1.9 surface VSG epitopes resulted from significant contributions by TI and TD mechanisms, whereas LouTat 1.8 infection selectively elicited TD responses. The RIA allowing separate quantitation of TI (nulnu) and TD plus TI (nul+) VSG surface epitope-specific B-cell responses during trypanosome infection is described in the legend to Fig. 1. The dashed lines represent the binding by pooled gamma globulin preparations obtained from each treatment group before trypanosome infection. INFECT. IMMUN. from LouTat 1-infected or immunized mice. The athymic and thymus-intact mice were able to mount VSG-specific B-cell responses to exposed epitopes after infection with LouTat 1 (Fig. 1B). The nul+ mice made somewhat stronger anti-vsg responses when infected, as indicated by the higher binding at the 10-2 serum dilution. Whether this represents a higher concentration of antibody or higher affinity is not clear from these data, although the similar slopes of these lines would indicate that these VSG-specific antibody populations have similar overall affinities but differ in concentration. The data also show that both TI and TD processes contribute to responses made to the native conformational form of the VSG surface coat during active infection. This can be seen by comparison of the curves within Fig. 1B. At the higher dilutions (e.g., 10-2) the TI sera showed no significant surface specific binding, whereas the infected nul+ mouse sera displayed maximal binding. This binding represents the TD enhancement of the native VSG surfacespecific B-cell responses, since the contribution by TI antibody binding has been diluted to background levels. Thus the TI and TD compartments of this response each independently display maximal binding in the RIA used, and therefore both probably represent significant components of the total native VSG surface-specific response in infected mice. Further dissection and quantitation of the TI and TD responses could not be made from these data; however, sera obtained from LouTat 1-infected nul+ mice (Fig. 1B) consistently displayed VSG surface epitope-specific binding activity equivalent to that of nulnu mouse sera assayed at 10-fold higher concentrations. Thus, the enhancement of the native VSG surface epitope-specific response in LouTat 1-infected mice by the presence of T cells may account for the production of the majority of this specific antibody population. Finally, it should be noted that the higher titer of antibody in LouTat 1-infected nul+ mice (relative to that of nulnu mice) was consistently observed during infection with other LouTat 1 serodeme variant antigenic types (Fig. 2). When the athymic nulnu and thymus-intact nul+ animals were immunized with purified VSG (500,ug) or paraformaldehyde-fixed trypanosomes (1010 cells), an anti-vsg surface epitope-specific response was detected only in the nul+ mice (Fig. 1C and D). The day 10 gamma globulin preparations from the immunized nude mice showed no binding activity greater than that of the normal serum controls (Fig. 2A) to the surface of live LouTat 1 trypanosomes. Immunization with various doses of these antigens also failed to elicit a surface epitope-specific immune response to viable LouTat 1 parasites in nude mice. In contrast, purified VSG 1 preparations or fixed LouTat 1 parasites elicited VSG surfacespecific responses in the thymus-intact nul+ littermate mice. These nul+ TD B-cell responses were weaker than those triggered by LouTat 1 infection, as shown by the lack of significant binding in the higher immunized serum dilutions (>10-2; Fig. 1C and D) as compared with the infected serum binding profiles (Fig. 1B). Thus, purified VSG (500,ug per mouse) or fixed trypanosome preparations (1010 cells per mouse) elicited TD native VSG surface epitope-specific B-cell responses, albeit less strongly than composite (TI plus TD) responses elicited during infection. It would therefore seem that these molecules present at least a subset of those VSG surface-specific epitopes displayed by the live LouTat 1 parasites. Differential B-cell responses to LouTat 1 serodeme variant antigenic types in nude mice. To further examine the TI and TD components of the aggregate VSG surface epitopespecific B-cell responses, BALB/c nulnu and nul+ mice were infected with four additional cloned variant antigenic types (VATs) of the LouTat 1 serodeme. Figure 2 shows the VSG surface-specific antibody binding results obtained from RIA analyses of day 10 serum samples from mice infected with clone LouTat 1.2, 1.5, 1.8, or 1.9. These additional T. brucei rhodesiense clones displayed immune response kinetics identical to that of LouTat 1 (see Discussion). As observed with LouTat 1 (Fig. 1), infection with these additional VATs triggered more vigorous VSG surface-specific B-cell responses in nul+ mice than in the athymic nulnu mice. A single exception was observed with LouTat 1.9- infected mice at a low serum dilution (i.e., 10-1). Analyses of sera obtained from LouTat 1.8-infected nulnu mice revealed that this T. brucei rhodesiense clone did not elicit a TI VSG surface epitope-specific B-cell response in nude mice. However, the infected thymus-intact nul+ littermate mice did produce antibody to VSG surface epitopes displayed on live LouTat 1.8 parasites. Repeated analyses of nulnu mouse sera collected at various times postinfection did not demon-

5 VOL. 58, 1990 strate VSG surface epitope-specific responses. Thus, infection with this particular clone of LouTat parasites selectively elicited only TD VSG surface-specific responses. Overall these data indicate that during the course of trypanosome infection TI and TD VSG surface epitope-specific B-cell responses occur to different trypanosomes (i.e., different VATs of the same serodeme: LouTat 1, 1.2, 1.5, and 1.9). However, there may be exceptions when only TD responses are made to exposed VSG epitopes (i.e., as with LouTat 1.8). The frequency of this phenomenon was one of five VATs in this study. However, only five VATs were examined. DISCUSSION The purpose of these studies was to examine the T-cell dependency of B-cell responses, specific for exposed epitopes of the VSG, that occur during active T. brucei rhodesiense infection. In addition, in these studies we examined B-cell responses to VSG epitopes after presentation of murine hosts with alternate forms of VSG (i.e., purified soluble VSG and VSG displayed on paraformaldehyde-fixed parasites). An RIA capable of selectively quantitating VSG surface epitope-specific antibody responses was used to analyze gamma globulin preparations derived from trypanosome-infected or VSG-immunized BALB/c nulnu and nul+ mice. Both substrains of mice were infected; gamma globulin was prepared after 7, 10, or 14 days and subjected to RIA analysis. A comparison of the kinetics and maximal specific antibody concentrations in sera (see Results) revealed that these responses in nulnu and nul+ mice were similar, with both substrains reaching maximal concentrations in serum on day 10. In addition, the affinity-purified 125I-labeled rabbit anti-mouse gamma globulin reagent was a carefully titrated mixture of anti--y and anti-,u antibodies (40:1), which detected IgG and IgM at approximately similar concentrations (based on IgG and IgM standards; see Materials and Methods). Finally, the low numbers of trypanosomes in infected nulnu and nul+ mice were also equivalent at day 10; the first systemic parasitemia was cleared by this time postinfection. This experimental design allowed an examination of the biologically relevant protective VSG-specific B-cell responses that occur in vivo during active infection. Results obtained were thus not complicated by binding of B-cell products to buried or sequestered VSG epitopes not normally displayed on the VSG surface coat of live parasites; the role of these nonprotective responses is not well understood, and they may be associated primarily with immunopathological reactions observed during infection (11, 22). Overall, the results demonstrate for the first time that VSG surface epitope-specific B-cell responses in infected hosts represent aggregate TI and TD responses, whereas the immunization procedures selectively triggered TD VSG surface-specific responses (see below). The infected athymic nulnu mice clearly made only TI surface VSG epitopespecific responses, whereas nul+ mice produced much stronger responses in the presence of T cells (Fig. 1; see Results). This difference in binding may be due to the production of higher concentrations of nul+ specific antibodies rather than higher-affinity antibodies as evidenced by the similar slope of the lines in Fig. 1B. Similar patterns (within error) of B-cell responses were also observed during infection with other VATs of the LouTat 1 serodeme (Fig. 2). Thus, in the presence of T cells a significantly stronger VSG surface epitope-specific response was mounted during active infection with LouTat 1, 1.2, 1.5, 1.8, and 1.9. TI AND TD VSG-SPECIFIC B-CELL RESPONSES 2341 Whether this T-cell augmentation has as its basis classical MHC restricted and antigen-processing cell-dependent stimulation of VSG-specific T-helper cells is clearly not addressed by data presented here and is currently under investigation. The T-cell-independent component of the VSG surface epitope-specific B-cell response was influenced by the mode of VSG presentation (or the particular infecting trypanosome clone, see below) to the host. In contrast, the TD antisurface responses were invariably observed regardless of the mode of presentation; immunization with purified VSG or paraformaldehyde-fixed trypanosomes triggered surface epitope-specific B-cell responses only in the thymus-intact nul+ mice. The athymic nulnu littermates did not develop surface-specific responses under these circumstances. Since the athymic mice did not respond, these particular immunization procedures must selectively stimulate TD responses. Such results probably indicate that specific responses in nul+ immunized animals result from classical major histocompatibility complex (MHC) restricted processing and presentation of monomeric VSG molecule determinants to VSG-specific TH cells. These responses would not be expected to occur in the nulnu animals. In addition, the immunized nul+ mouse anti-surface VSG responses were significantly weaker than those elicited during infection (as compared with responses of nul+ infected mice; Fig. 1B), as evidenced by the lack of binding at the higher serum dilutions. Thus, purified VSG molecules or paraformaldehydefixed trypanosomes must expose or present only certain of the surface epitopes and trigger only TD VSG surface epitope-specific B-cell responses. Indeed, the weak nature of the anti-surface B-cell responses elicited by the fixed parasites may indicate that this immunogen poorly presents native surface epitopes or presents only a small fraction of all epitopes, displayed on the live trypanosome surface VSG. However, the distinction between infection and immunization is not trivial; parasite-derived factors may influence immune responsiveness in many ways. The infected host surface VSG epitope-specific B-cell responses clearly represent aggregate TI and TD responses, and both of these mechanisms are responsible for the production of a significant fraction of the specific antibody produced. An exception to this general trend was noted during RIA analysis of gamma globulin obtained from Lou- Tat 1.8-infected mice. Infection with this particular trypanosome clone resulted in the production of VSG surface epitope-specific antibody exclusively by TD mechanisms. This was shown by the inability of the LouTat 1.8-infected nulnu mice to produce any anti-vsg 1.8 antibodies that reacted with exposed epitopes (Fig. 2C). These data collectively demonstrate that TI and TD VSG surface epitopespecific B-cell responses are made to most (four of five) trypanosome variants that arise during relapsing parasitemias initiated by LouTat 1; however, growth of certain clones (i.e., LouTat 1.8) may result in the production of VSG-specific antibodies by TD mechanisms alone. Whether this phenomenon results from some significant conformational alteration in the surface coat, lack of an appropriate second signal to B cells, parasite-specific immunosuppression, or "holes" in the TI B-cell repertoire currently is under investigation. Finally, differences in the course of infection in the nulnu and nu/+ mice may be important also. Other studies involving trypanosome infection of nude mice (6, 14, 21) or B-cell responses to surface VSG epitopes (2, 6, 13, 15, 16, 22) have not directly examined the T-cell dependency of these immune responses. Various experimen-

6 2342 REINITZ AND MANSFIELD tal systems and species of trypanosomes have been used, making comparisons difficult or inconclusive; however, the data presented here are in general agreement with previous studies. For example, athymic (nulnu) and thymus-intact (normal) C57BL/6 mice infected with T. congolense produced similar peak anti-surface VSG epitope-specific antibody titers when examined in a complement-mediated lysis assay (17). Further, the kinetics of these responses were significantly different; thymus-intact mice made peak responses earlier (day 6) and maintained these levels longer (through day 14). These careful studies also showed that the assay of Pinder et al. was much more sensitive to the mu immunoglobulin isotype (the only isotype produced by nulnu mice), owing to its highly efficient rate of complement fixation. Although this assay is difficult to directly compare with the present study, and since species differences may exist (T. brucei rhodesiense versus T. congolense), it was clear the T. congolense-specific B-cell responses to surface VSG epitopes were modulated by the presence of T cells in the thymus-intact mice. Thus, these immune responses probably also represent aggregate TI and TD responses. Finally, studies from this laboratory (19; C. M. Theodos and J. M. Mansfield, J. Immunol., in press; C. M. Theodos, D. M. Reinitz, and J. M. Mansfield, J. Immunol., in press) and others (2, 15, 16) have examined monoclonal or polyclonal antibody binding to parasite VSG. In general, these experiments were not intended to examine the role of T cells, yet they have indirectly shown such involvement by induction of hybridoma VSG-specific IgG antibodies. Thus, B-cell responses specific for VSG surface-exposed epitopes on the LouTat serodeme of T. brucei rhodesiense, and probably other trypanosome species and subspecies, represent aggregate TI and TD processes. Inasmuch as there was little data directly addressing such T-cell dependency, more basic information was needed before T-cell epitope mapping studies of the VSG molecule. The present study provides some of this necessary information, which serves as a base for our current investigations of B- and T-cell epitopes detectable on the VSG molecule and the means by which immune responses to them are regulated (Theodos and Mansfield, in press; Theodos et al., in press). ACKNOWLEDGMENTS This work was supported by Public Health Service grant AI from the National Institutes of Health to John M. Mansfield and by Public Health Service postdoctoral award AI from the National Institutes of Health to David M. Reinitz. LITERATURE CITED 1. Campbell, G. H., K. M. Esser, and M. 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