Abstract
A weakness of the hu-PBL–SCID model for the study of human immune functions is the appearance of anergy and the consequent loss of T cell function. We demonstrate here that human T cells retain normal functions during the early stage of chimerism. At 1 and 2 weeks post-engraftment, T cells isolated from the peritoneal cavity of hu-PBL chimeras could be activated and proliferated upon stimulation with phytohaemagglutinin (PHA) or specific antigens to which the cell donor was known to be immune. T cells derived from hu-PBL–SCID and hu-PBL–NOD/LtSz-scid (NOD/SCID) mice not only proliferated but also produced interferon-gamma (IFN-γ) and IL-5 following in vitro stimulation with tetanus toxoid (TT) or hepatitis B surface antigen (HBsAg). These antigen-specific T cells could only be demonstrated when cognate antigen was administered together with or immediately following the PBL transfer. Without an early rechallenge with antigen in vivo, no TT- or HBsAg-specific T cell responses could be elicited, showing the vulnerability and antigen-dependence of the T cell response. Vigorous anti-TT or anti-HBs responses could be observed in all chimeras. Administration of antigen together with the PBL graft enhanced the humoral anti-TT response in SCID and NOD/SCID mice but had little effect on the anti-HBs antibody response in NOD/SCID mice. These data confirm the observation that the B cell compartment in hu-PBL–SCID chimera is largely antigen-independent and extend this to SCID/NOD.
Keywords: T cell, hu-PBL–mouse chimera, TMβ1
INTRODUCTION
Severe combined immunodeficient (SCID) mice lack functional T and B lymphocytes due to defective recombination of lymphocyte antigen receptor genes, and allow functional engraftment of allogeneic or xenogeneic cells [1]. Xenogeneic transplantation of human lymphoid cells into SCID mice provides a useful model for the investigation of a variety of problems in human immunology, including the study of the normal human immune function, human vaccine development and the pathogenesis of human viral diseases such as HIV [2,3]. SCID mice reconstituted with human PBL showed spontaneous secretion of human immunoglobulin and a specific human antibody response was induced following immunization with tetanus toxoid (TT) [4,5]. The production of human immunoglobulins was used as a marker of successful reconstitution.
Several laboratories [6–14] have investigated human T cell functions in human PBL–SCID mice. Tary-Lehmann et al. [13] showed that human T cells, freshly isolated from long-term hu-PBL–SCID chimeras, were refractory to stimulation by anti-CD3 or phytohaemagglutinin (PHA) and were in a state of anergy. Attempts by several other groups to demonstrate antigen-specific proliferation or cytotoxic T cell function in hu-PBL–SCID chimeras, 3–8 weeks after human PBL transfer, were generally unsuccessful [13]. The specific selection and expansion of few clones in these long-term chimeras induces a very restricted T cell repertoire [10]. Only a few reports showed antigen-specific cytotoxic T lymphocytes in hu-PBL–SCID chimeric mice [6,14–16].
At the early stage of human PBL engraftment very low numbers (0·1% of the original graft) of human T cells can be detected in hu-PBL–SCID chimera [9,17]. This makes the analysis of antigen-specific cellular immune responses extremely difficult. Different strategies have been applied to improve the engraftment of hu-PBL in SCID mice. Some investigators have increased the numbers of cells transferred [4,12]. This approach has only marginally improved the engraftment of human cells. Others have used different immunocompromised mouse strains such as NOD-SCID mice or SCID-Bg [18–22], which provide a new and improved host for the hu-PBL grafts compared with SCID mice. The pretreatment of the recipient mice with low-dose total body radiation [23–25] or the depletion of mouse natural killer (NK) cells by antibodies such as anti-asialo-GM1 [6,23,24] or anti-NK1.1 [22] improved the survival and expansion of the human cell graft. Recently, we [21,26] have shown that pretreatment of SCID and NOD/SCID mice with TMβ1 [27], a rat monoclonal antibody directed against the mouse IL-2 receptor β-chain, greatly improved the survival of human PBL. An additional total body radiation (3 Gy) further improved the survival of the human cells [21]. To develop a mouse model for the study of human vaccines and the pathogenesis of human viral diseases, we investigated the function of human T cells during the early stage of engraftment in irradiated and TMβ1-pretreated SCID and NOD/SCID mice.
MATERIALS AND METHODS
Mice
Homozygous C.B-17 scid/scid (SCID) mice or NOD/LtSz-scid (NOD/SCID) were bred and maintained under specific pathogen-free conditions and were used in these studies at an age of 10–12 weeks. One day before reconstitution with human PBL, mice were irradiated (3 Gy, gamma irradiation) and injected intraperitoneally with 1 mg TMβ1 [26,27] in 0·5 ml PBS to reduce endogenous mouse NK cell activity. All manipulations were performed by experienced hands and the study protocol was approved by the local animal ethics committee.
Engraftment of mice with human PBL and in vivo immunization
Human PBL, derived from buffy coats from healthy donors or heparinized venous blood from selected hepatitis B surface antigen (HBsAg)-vaccinated donors, were isolated by Ficoll–Hypaque (density = 1·077 g/ml; Nycomed Pharma, Oslo, Norway) centrifugation and injected intraperitoneally (1–2 × 107 hu-PBL per mouse in 0·5 ml PBS) into the recipient mice. For in vivo immunization with TT (obtained from Statens Seruminstitut, WHO, Copenhagen, Denmark), TT was injected intraperitoneally (10 μg/mouse) together with human PBL. For in vivo immunization with HBsAg, 2 μg of a commercial HBsAg vaccine (Engerix-B; SmithKline Beecham Biologicals, Brussels, Belgium) were injected subcutaneously into a hind leg 1 day after cell transfer.
Cell collection from the human–mouse chimeras and flow cytometric analysis
At days 7 and 14 following cell transfer, mice were bled and subsequently killed (three mice per group) by cervical dislocation. Peritoneal exudate cells (PEC) were obtained by two rounds of peritoneal lavage with 5 ml ice-cold PBS. PEC from three animals of the same experimental group were pooled. Viable mononuclear cells (propidium iodide-negative) in those pooled PEC suspensions were analysed by flow cytometry. To avoid non-specific staining, the murine cells were gated out with cytochrome-conjugated anti-mouse common leucocyte antigen CD45 (30-F11; PharMingen, Hamburg, Germany). Human cells in the PEC were characterized with the following MoAbs: CD45, CD3, CD4, CD8, CD14, CD19, CD45RO, CD25, CD69, CD71, HLA-DR, CD86. Antibodies were conjugated with FITC or PE. All MoAbs were purchased from Becton Dickinson (San Jose, CA) except for CD71 and HLA-DR, which were from Caltag Labs (San Francisco, CA). Cells (1 × 105) were incubated with antibodies on ice for 30 min, washed and resuspended in PBS containing 1% bovine serum albumin (BSA) and sodium azide (0·09%). At least 5000 cells were analysed on a FACScan (Becton Dickinson). Isotypically matched negative control antibodies were always used.
Lymphoproliferation and cytokine assays
For functional analysis PEC derived from animals of the same experimental group were pooled and fractionated by Ficoll–Hypaque centrifugation. The cells harvested from the interphase were further examined. For proliferation assays the cells (1 × 105 cells/well) were suspended in 200 μl complete RPMI 1640 medium. This consisted of RPMI 1640 supplemented with 25 mm HEPES, 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mm l-glutamine (all from Gibco, Grand Island, NY), 5 × 10−5 mβ-mercaptoethanol (Sigma Chemical Co., St Louis, MO) and 10% heat-inactivated human AB+ serum. The cells were cultured for 4 days in the presence of 5 × 104 irradiated (30 Gy, 60Co source), T-depleted (by CD2-coated Dynabeads; Dynal AS, Oslo, Norway) autologous PBL. These cultures were stimulated with TT (4 μg/ml; Statens Seruminstut), rHBsAg (3 μg/ml, recombinant, yeast-derived HBV envelope major protein, a gift from SmithKline Beecham Biologicals), PHA (3 μg/ml; Sigma), or were left without stimulating antigen (control culture). The quality of the T-depleted PBL was examined by flow cytometry and T cell content was always <2%. All cultures were performed in triplicate. 3H-thymidine (0·5 μCi/well) was added 18 h before the cultures were harvested using an automatic cell harvester. The incorporation of 3H-thymidine into dividing cells was measured in a liquid scintillation counter (LKB-Wallac 8100 counter; LKB, Bromma, Sweden). The data were expressed as the mean counts of triplicate determinations and stimulation index (SI) calculated as: SI = mean ct/min of antigen-stimulated cultures/mean ct/min of control cultures.
Supernatants from the proliferation assays were collected at 72 h and kept frozen at −20°C until tested. Commercial kits were used to determine human interferon-gamma (IFN-γ) (MEDGENIX IFN-γ EASIATM kit; BioSource Europe S.A., Nivelles, Belgium) and IL-5 (Genzyme Human Interleukin-5 Kit). The assays were performed according to the manufacturer's guidelines.
Determination of total human IgG, IgM and antigen-specific antibody
Total human IgG and IgM concentrations in chimeric mouse plasma were determined by an in-house ELISA as described previously [26]. The concentration of human TT-specific IgG in human and mouse plasma was measured using the Tetanus Toxoid Sensitive IgG Antibody Kit (Gamma S.A. Angleur-Liége, Belgium). This method has a detection limit of 50 U/l. HBs antibodies (anti-HBs) were measured with ETI-AB-AUK-3 (anti-HBs enzyme immunoassay from DiaSorin Biomedica, Saluggia, Italy). Titres were expressed as U/l.
Statistical analysis
Comparisons between groups were done using Mann–Whitney U-test. The Statistical Package SPSS 7.5 (SPSS Inc., Chicago, IL) was employed.
RESULTS
Phenotypic characteristics of T cells recovered from chimeric mice
PBL derived from one buffy coat were transferred into TMβ1-pretreated and irradiated SCID mice (n = 6). Similarly, PBL from another buffy coat were transferred into pretreated (TMβ1 + irradiation) NOD/SCID mice (n = 6). One and 2 weeks after cell transfer the animals (three per time point) were killed and the human leucocyte content and subset distribution in pooled PEC suspensions were analysed by flow cytometry. The relative T cell content of PEC in SCID and NOD/SCID mice were 79% and 81% after 1 week and 89% and 78% after 2 weeks, respectively. The expression of activation markers on these T cells was also assessed and is shown in Table 1. The expression of CD45RO on the T cells obtained 1 and 2 weeks following engraftment was clearly higher than in fresh PBL (generally <50%). Other activation markers, HLA-DR, CD25, CD71, could also be found on a fraction of the T cells 1 and 2 weeks after transplantation. The expression of these markers was higher at week 1 than at week 2. Since the cells used for the transplantation of SCID and NOD/SCID mice were derived from different donors, the distribution of the activation markers on the T cells in these two animal strains cannot be compared.
Table 1.
Expression of activation markers on peritoneal exudate cells (PEC) from the chimeras at 1 and 2 weeks post-engraftment
| PEC (%) | Percent of activation markers on human CD3+ T cells | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Donor | Mice | CD45 | CD3 | CD19 | HLA-DR | CD45RO | CD86 | CD25 | CD71 | CD69 | |
| Day 0 | 98 | 54 | 19 | 0·7 | 37·6 | 1·7 | 5·1 | 0·9 | 0 | ||
| A | SCID | Day 7 (n = 3) | 85 | 79 | 15 | 51·7 | 73·1 | 29 | 13·3 | 38·6 | 0·02 |
| Day 14 (n = 3) | 93 | 89 | 4 | 12·7 | 48·5 | 12 | 11·7 | 18·98 | 0·5 | ||
| Day 0 | 97·5 | 46 | 6·6 | ND* | ND | ND | ND | ND | ND | ||
| B | NOD/SCID | Day 7 (n = 3) | 90 | 81 | 2·3 | 37·7 | 95·8 | 43 | 63·8 | 72·3 | 3·5 |
| Day 14 (n = 3) | 54 | 78 | 1·5 | 8·34 | 83·2 | 13 | 43·6 | 14·9 | 1·6 | ||
Not determined.
Human PBL derived from two buffy coats (donors A and B) were transferred intraperitoneally into the pretreated SCID and NOD/SCID mice, respectively. At day 7 and day 14 following cell transfer, mice (three mice/per group) were killed. The percentage of human cells in the pooled PEC that were derived from the same experimental group and the expression of different activation markers on human T cells were determined by flow cytometry with dual-label analysis as described in Materials and methods.
HBsAg-specific memory T cell response in hu-PBL–NOD/SCID mice
To examine the HBsAg-specific T cell response in PEC from NOD/SCID transplanted with PBL from an HBsAg-immune donor, we transferred PBL (1 × 107 cells/mouse) into (TMβ1 + irradiation) pretreated NOD/SCID (n = 12). Six mice were immunized with 2 μg HBsAg the day after cell transfer, while the remaining animals did not receive antigen. One and 2 weeks after the inoculation with human PBL three animals of each group were killed, the PEC were recovered and stimulated in vitro with HBsAg or PHA as described. Both HBsAg-immunized and non-immunized chimeric NOD/SCID mice displayed PHA-induced responses at weeks 1 and 2. Only in the HBsAg-immunized animals was an HBsAg-specific proliferative response observed at week 1 and week 2 (Table 2). When animals were pretreated with irradiation or TMβ1 separately, no HBsAg-specific proliferative responses could be measured, irrespective of the administration of HBsAg at the time of transplantation (data not shown).
Table 2.
Hepatitis B surface antigen (HBsAg)-specific proliferative responses of peritoneal exudate cells (PEC) from the chimeras at 1 and 2 weeks post-engraftment
| HBsAg-immunized NOD/SCID | Non-immunized NOD/SCID | ||||
|---|---|---|---|---|---|
| PEC harvested on | In vitro stimulation | Ct/min mean ±s.d. | SI | Ct/min mean ±s.d. | SI |
| Week 1 | (n = 3) | (n = 3) | |||
| HBsAg (3 μg/ml) | 4725 ± 995 | 2·6 | 3006 ± 926 | 0·9 | |
| PHA (3 μg/ml) | 26 816 ± 2605 | 14·6 | 24 776 ± 434 | 7·7 | |
| Blank | 1834 ± 470 3 | 210 ± 438 | |||
| Week 2 | (n = 3) | (n = 3) | |||
| HBsAg (3 μg/ml) | 2945 ± 1217 | 8·8 | 653 ± 33 | 1·7 | |
| PHA (3 μg/ml) | 10 667 ± 3253 | 32 | 7936 ± 1253 | 21 | |
| Blank | 334 ± 48 | 374 ± 75 | |||
PBL (1 × 107 cells/mouse) from an HBsAg-immune donor were transferred intraperitoneally into the pretreated NOD/SCID mice (n = 12). Six mice were immunized subcutaneously with 2 μg HBsAg the day after cell transfer, the other six mice received PBS as control. One and 2 weeks after engraftment three mice from each group were killed. PEC were collected, stimulated in vitro with HBsAg or phytohaemagglutinin (PHA), or left without stimulating antigen. The detailed data are shown as mean counts of triplicate cultures and as stimulation index (SI).
TT-specific memory T cell immune response in human PBL–NOD/SCID or SCID mice
Human PBL (2 × 107 cells/mouse) isolated from one buffy coat were injected intraperitoneally into 12 NOD/SCID mice, which were pretreated with TMβ1 and radiation. Similarly, PBL derived from another buffy coat were injected into 12 SCID mice. In six SCID mice and six NOD/SCID mice, TT (10 μg/mouse) was administrated intraperitoneally at the time of human PBL transfer. The remaining SCID and NOD/SCID mice were given PBS as control. After 1 and 2 weeks, three mice from each experimental group were killed and their PEC were pooled and run over a density gradient. Triplicate cultures of fractionated PEC (1 × 105 cells/well) were stimulated with TT (4 μg/ml) or left unstimulated. Table 3 shows that the PEC isolated after 1 or 2 weeks from the TT-immunized SCID/NOD and SCID mice strongly proliferated upon in vitro restimulation with TT. Such a vigorous proliferation was not observed in the PEC derived from non-immunized NOD/SCID or SCID mice. PEC derived from SCID mice, 2 weeks after the transfer of hu-PBL from donor 2, displayed a high spontaneous proliferation and a marked response towards TT (SI = 6). This phenomenon remains unexplained.
Table 3.
Tetanus toxoid (TT)-specific proliferative responses of peritoneal exudate cells (PEC) from the chimeras at 1 and 2 weeks after hu-PBL engraftment
| TT-immunized mice | Non-immunized mice | |||||
|---|---|---|---|---|---|---|
| Donor and mice | PEC harvested on | In vitro stimulation | Ct/min mean ±s.d. | SI | Ct/min mean ±s.d. | SI |
| (n = 3) | (n = 3) | |||||
| Donor 1 in NOD/SCID | Week 1 | TT (4 μg/ml) | 52 276 ± 29 | 29 | 1239 ± 115 | 2·4 |
| Blank | 1794 ± 306 (n = 3) | 1 | 514 ± 208 (n = 3) | 1 | ||
| Week 2 | TT (4 μg/ml) | 8033 ± 2800 | 15 | 1679 ± 669 | 1·7 | |
| Blank | 534 ± 105 | 1 | 994 ± 342 | 1 | ||
| Donor 2 in SCID | (n = 3) | (n = 3) | ||||
| Week 1 | TT (4 μg/ml) | 24 298 ± 2766 | 19·1 | 3683 ± 653 | 1·7 | |
| Blank | 1270 ± 297 (n = 3) | 1 | 2145 ± 493 (n = 3) | 1 | ||
| Week 2 | TT (4 μg/ml) | 69 434 ± 7552 | 31·7 | 38 656 ± 2981 | 6 | |
| Blank | 2193 ± 929 | 1 | 6445 ± 1589 | 1 | ||
PBL (2 × 107 cells/mouse) derived from two buffy coats (donors 1 and 2) were transferred intraperitoneally into the pretreated NOD/SCID (n = 12) and SCID (n = 12) mice, respectively. TT (10 μg/mouse) was injected intraperitoneally together with PBL in six NOD/SCID and six SCID mice; the remaining mice were given PBS as control. One and 2 weeks after PBL inoculation, PEC from the same experimental group of mice (three mice per group) were collected, and TT-specific lymphoproliferation assays were performed as described. Detailed data are expressed as mean counts of triplicate cultures and as stimulation index (SI).
Cytokine production of human lymphocytes freshly isolated from the chimeric mice in response to in vitro stimulation with recall antigen (TT)
To assess the ability of both SCID and NOD/SCID mice to be reconstituted by human PBL and to evaluate TT-specific proliferation and cytokine secretion of human T cells, both NOD/SCID (n = 6) and SCID (n = 6) mice, pretreated with TMβ1 and radiation, were inoculated intraperitoneally with PBL (2 × 107 cells/mouse) from a single buffy coat. Half of the SCID and NOD/SCID mice were given TT (10 μg/mouse, intraperitoneally) together with the hu-PBL. The control group received PBS. The proliferation and cytokine production (human IL-5 and IFN-γ) of PEC, harvested 2 weeks after human PBL engraftment, were determined. The PEC from both chimeric SCID mice and NOD/SCID mice, which were immunized with TT at the time of human cell transfer, proliferated and produced human IL-5 and IFN-γ in response to an in vitro stimulation with TT. The data in Table 4 demonstrate that the proliferative and cytokine responses of the cells derived from SCID and NOD/SCID do not differ substantially. The PEC derived from NOD/SCID mice that were not exposed to TT at the time of PBL transfer did not display a proliferative or cytokine response upon in vitro culture with TT. As in previous experiments (Table 3) the PEC derived from chimeric SCID mice that were not immunized with TT in vivo were responsive to in vitro stimulation with TT. However, the observed responses (proliferative response with thymidine incorporation of 15 348 ct/min and cytokine production with IFN-γ = 7·7 U/ml and IL-5 = 5·6 pg/ml), were considerably lower than in the immunized animals.
Table 4.
Antigen-specific proliferation and cytokine production of peritoneal exudate cells (PEC) from the chimeras at 2 weeks after hu-PBL transfer
| TT-immunized chimeras | Non-immunized chimeras | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| In vitro stimulation | Ct/min mean ±s.d. | SI | IFN-γ (U/ml) | IL-5 (pg/ml) | Ct/min mean ±s.d. | SI | IFN-γ (U/ml) | IL-5 (pg/ml) | |
| NOD/SCID | TT (4 μg/ml) | 17 447 ± 699 | 37 | 74 | 101·1 | 3361 ± 52 | 1·5 | 7·9 | 0 |
| (n = 6) | Blank | 471 ± 73 | 0·95 | 0 | 2255 ± 52 | 1 | 7·3 | 0 | |
| SCID | TT (4 μg/ml) | 44 177 ± 6863 | 25·5 | 78 | 323·8 | 15 348 ± 1614 | 27 | 7·7 | 5·6 |
| (n = 6) | Blank | 1733 ± 136 | 1 | 0·7 | 0 | 564 ± 189 | 1 | 0·5 | 0 |
PBL (2 × 107 cells/mouse) derived from the same buffy coat were transferred intraperitoneally into the pretreated SCID (n = 6) and NOD/SCID (n = 6) mice. Tetanus toxoid (TT; 10 μg/mouse) was injected intraperitoneally together with PBL into three NOD/SCID and three SCID mice; the remaining mice received PBS as control. Two weeks later, PEC from the same experimental group were recovered and TT-specific proliferation assays were performed. Detailed data are expressed as mean counts of triplicate cultures and as stimulation index (SI). The supernatants from these assays were collected at 72 h and kept frozen at −20°C until tested for human IFN-γ (U/ml) and IL-5 (pg/ml).
Human immunoglobulin production in the chimeric mice
In all the experiments shown before, the concentration of human immunoglobulins (IgM, IgG) in mouse plasma was measured at week 1 and/or week 2. The in vivo administration of HBsAg or TT had no effect on the total human immunoglobulin levels (P > 0·1). Data from two representative experiments are shown in Table 5. Data in SCID and NOD/SCID cannot be compared since the cells used for the transplantation of SCID and NOD/SCID were derived from different donors.
Table 5.
Total human IgG, IgM and tetanus toxoid (TT)-specific antibody production in human PBL–SCID and NOD/SCID mice
| Mice | Immunization | Total IgG (ng/ml) | Total IgM (ng/ml) | Anti-TT immunoglobulin (U/l) | |
|---|---|---|---|---|---|
| Donor 1 in NOD/SCID | |||||
| Week 1 | NOD/SCID | PBS | 49 091 ± 10 933 | 2547 ± 522 | 508 ± 695 |
| NOD/SCID | TT | 42 472 ± 11 602 | 1948 ± 598 | 247 ± 328 | |
| Week 2 | NOD/SCID | PBS | 2022 717 ± 569 539 | 67 145 ± 18 736 | 40 799 ± 25 987 |
| NOD/SCID | TT | 2605 548 ± 761 251 | 54 894 ± 13 597 | > 500 000 | |
| Donor 2 in SCID | |||||
| Week 1 | SCID | PBS | 26 674 ± 6878 | 44 181 ± 10 507 | 70 ± 26 |
| SCID | TT | 43 442 ± 14 209 | 71 646 ± 24 879 | 2138 ± 376** | |
| Week 2 | SCID | PBS | 786 002 ± 211 104 | 188 371 ± 49 457 | 3285 ± 471 |
| SCID | TT | 1010 082 ± 176 170 | 205 377 ± 135 933 | 47 224 ± 17 937** | |
The concentration of human IgM and IgG and antigen-specific antibody were determined in each mouse plasma at weeks 1 and 2 as described in Materials and methods.
Significant differences (P < 0·05) between the immunized (TT) and non-immunized (PBS) animals.
Antigen-specific antibody responses in the chimeric mice
Antigen-specific humoral immune responses (anti-HBs antibody, anti-TT antibody) at week 1 and week 2 were measured in the plasma of all animals included in the above mentioned experiments. As shown in Table 5 (data from the same experiment of Table 3), anti-TT antibody production in TT-immunized hu-PBL–SCID at week 1 and week 2 was 2138 U/l and 47 224 U/l, which was 30 times and 14 times higher than in non-immunized hu-PBL–SCID mice (70 U/l at week 1, 3285 U/l at week 2), respectively (P < 0·05). At week 2 the anti-TT titres measured in NOD/SCID mice were more than 10 times higher in the TT-immunized mice than in the non-immunized mice. This effect was not observed at week 1. Anti-HBs responses in hu-PBL–NOD/SCID mice (experiment shown in Table 2) were also measured in both HBsAg-immunized and non-immunized mice: 878 ± 95 U/l and 1049 ± 872 U/l at week 1, 62 798 ± 23 833 U/l and 45 162 ± 1063 U/l at week 2, respectively. The difference in anti-HBs antibody production between HBsAg-immunized and non-immunized hu-PBL–NOD/SCID mice was not significant (P > 0·1).
DISCUSSION
A hu-PBL–SCID mouse becomes a useful tool to study human immune functions when sufficient numbers of human lymphocytes survive for extended periods in a normal functional state. We and others have shown that the hu-PBL–SCID model is a powerful tool to examine B cell responses ([21,26] and manuscript in preparation). The study of human T cell responses in the in vivo environment of the SCID mouse is hampered by the appearance of anergy and the consequent loss of T cell function [9,10,12,13] and the rapid disappearance of T cells from the peritoneal cavity when PBL are introduced in that compartment [9,17].
We recently designed a conditioning regimen that strongly improved the survival of human cells transferred into SCID and NOD/SCID mice. This method is based on the combined use of total body irradiation (3 Gy) and a rat antibody against the mouse IL-2 receptor β-chain (TMβ1) [21,26]. The strong and rapid engraftment of human PBL thus obtained enabled us to study the phenotypic and functional characteristics of human T cells recovered from chimeras reconstituted with PBL from TT- and HBsAg-immune donors.
The analysis of the PEC harvested 1 and 2 weeks after the transfer of human PBL into the peritoneal cavity of SCID and NOD/SCID mice showed that over 80% of the recovered human cells were T lymphocytes, most of which expressed CD45RO and could thus be considered as memory T cells. Similar observations were made by other investigators exploring hu-PBL–SCID models [10,12]. Further analysis of these human memory T cells showed their expression of multiple activation markers such as HLA-DR, CD25, CD69 and CD71. The expression of these markers was higher on week 1 than on week 2. This is surprising considering the common view that human T cells in chimeras are continuously stimulated by xenoantigen, a phenomenon which progressively shapes the human T cell repertoire and ultimately leads to anergy and loss of T cell function [9,10,12,13]. Whether the observed decline in activation markers is due to a dissemination of activated T cells from the peritoneum to other organs or to a decline in the expression of these markers remains unclear at present.
In long-term human PBL–SCID chimera the human T cells reach an anergic state and become refractory to specific as well as to polyclonal, mitogenic stimuli [3,9,13]. The PEC recovered from our chimeras proliferated upon in vitro stimulation with PHA, ruling out an anergic state. Proliferative T cell responses to certain recall antigens against which the cell donor was sensitized (e.g. TT, diphteria toxin, PPD) [13] and specific CTL responses to antigens from defined pathogens (e.g. nucleoprotein from influenza A virus) [6] have been observed in the early phase of chimerism. In these experiments irradiated, autologous PBL were used as a source of antigen-presenting cells (APC) to restimulate the chimeric T cells. Although such feeder cells were unable to proliferate they could still secrete IL-2 upon stimulation with the recall antigen and thus induce bystander proliferation in the chimera-derived human T cells [3]. Experiments like these should be interpreted with care and are no absolute proof of the antigen-specificity of the observed T cell response. The human PBL used as APC in our experiments were T cell-depleted (T cell content <2%), which excludes bystander proliferation and favours the antigen-specific character of the proliferation.
To examine antigen-specific T cell function, PBL from TT- or HBsAg-immune donors were transferred in the peritoneal cavities of SCID or NOD/SCID mice. When TT or HBsAg were administered concomitantly or within 24 h after cell transfer, the T cells recovered from the peritoneum at week 1 and week 2 displayed vigorous antigen-specific proliferative responses and secreted substantial amounts of IFN-γ and IL-5. When no antigen was administered at the time of cell transfer the ensuing proliferative responses were weak or absent and no cytokine production could be measured. These data clearly show the need of T cells for an early antigen exposure in vivo in order to be stimulated and rescued from early cell death.
We recently found that the mouse strain and the conditioning regimen used to generate hu-PBL chimeras determine the magnitude and the antigen independence of the B cell responses towards recall antigens [21]. This observation is confirmed and extended here. Spontaneous, meaning without re-exposure to cognate antigen in vivo, antigen-specific antibody responses towards TT and HBsAg were observed in SCID (TT) and NOD/SCID (TT and HBsAg) mice. While the addition of TT to PBL upon i.p. cell transfer enhanced the anti-TT response when compared with that of PBL that were administered in the absence of TT, the anti-HBs response seemed not to benefit from such an antigen re-exposure in NOD/SCID mice. It is tempting to speculate that TT-specific T cells contribute to this enhanced response. However, it remains unclear why in NOD/SCID mice the HBsAg-specific T cells, the presence of which has clearly been demonstrated, are not contributing to the anti-HBs response in a similar fashion. The difference between TT- and HBsAg-specific responses may reside in the nature of the antigens (soluble protein TT versus particulate antigen HBsAg) or in their way of administration (admixed to the transferred PBL for TT versus subcutaneous injection after 24 h for HBsAg).
In conclusion, the data shown here demonstrate for the first time the presence of antigen-specific T cells in hu-PBL–NOD/SCID chimeras. When examined within 2 weeks following cell transfer, human T cells retain antigen specificity and are able to proliferate and produce cytokines upon stimulation with recall antigens in vitro. While exposure to cognate antigen in vivo is essential to safeguard this specific T cell function, the B cell compartment appears largely antigen-independent. Differences in the TT-specific antibody responses in chimeras that were or were not exposed to the antigen in vivo suggest that TT-specific T cells may contribute to the magnitude of the specific antibody responses.
Acknowledgments
The authors thank Dr Tom Boterberg (Department of Experimental Cancerology, University Hospital, Ghent) for the irradiation of the mice. T.C. is supported by the grant of the Ministry of Education of People's Republic of China. Part of this work was supported by the fund from Scientific Research-Flanders (NR: G.022.00).
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