Introduction

Severe combined immunodeficiency (SCID) mice have a genetic defect that results in the inability to rearrange T cell receptor and immunoglobulin genes, and produce immunocompetent T cells and B cells, respectively.¹ These mice lack a functional immune system and have proved to be suitable hosts for the engraftment of human haematopoietic stem cells and mature PBL.^{2, 3} The functional capacity of the engrafted human immune cells and their potential role in generating humoral and cellular immune responses in vivo, has been investigated. Long-term B cell antibody production, including the production of xenoreactive antibodies,⁴ specific antibodies to recall antigen,⁵ and auto-antibody production,⁶ has been demonstrated in SCID mice repopulated with human PBL. In the case of T cells, those isolated from Hu-PBL-SCID chimeras appeared to be in a state of unresponsiveness to conventional T cell stimulation, although this was reversible.⁷ More recent studies have indicated that rejection of islet allografts in the Hu-PBL-SCID recipient was mediated by functionally competent donor graft infiltrating T cells.⁸ In addition, anti-tumour immunity in Hu-PBL-SCID to the development of Daudi lymphoma has been shown to involve T cell proliferation in response to Daudi.⁹ Together, these findings indicate that the function of the human immune system is retained, at least partially, in PBL-reconstituted SCID mice. To this end, human-SCID chimeric animal models have been used to study a variety of human immune responses, including those elicited in response to infection,¹⁰ self-antigens,⁶ tumours,⁹ and allografts.⁸

The successful engraftment of human immunocompetent cells into SCID mice is variable and may be attributed to a number of factors including host NK activity,¹¹ donor EBV status,³ and the activation status of the cells to be transferred.¹² Activation of human PBL with an anti-CD3 monoclonal antibody has been shown to increase the incidence of engraftment of human PBL into SCID lymphoid tissues, and resulted in T cell engraftment within the thymus.¹² In vitro pre-activation of human T cells by mixed lymphocyte tissue culture¹³ and human growth hormone,¹⁴ have also been shown to enhance the migration of human T cells to SCID lymphoid tissues following reconstitution. These studies have largely utilized FACS analysis of in vitro isolated cell populations to determine the levels of human cell engraftment in SCID mice.

In the present study we have compared the engraftment of fresh unstimulated human PBL and anti-CD3-stimulated PBL in SCID mice. Engraftment was assessed in SCID mice by measurement of human IgG (HIgG) in mouse sera, detection of human-specific HLA-DR DNA in SCID periphery, and, in situ, by immunohistochemical staining of mouse tissues with anti-human CD45 and CD3 antibodies.

Materials and methods

Mice

Approximately 8-week-old male C.B-17 scid/scid (SCID) mice were obtained from a breeding colony (Walter and Eliza Hall Institute, Melbourne, Victoria, Australia). Mice were housed under sterile conditions at the Princess Alexandra Hospital animal facility for the duration of the experiment. All mice were 'non-leaky' SCID and had mouse IgG levels < 2 g/mL as assessed by inhibition ELISA.

Preparation of PBL and SCID reconstitution

Approximately 100 mL peripheral blood was obtained each from two adult male healthy donors (donor A and donor B). Both donors were EBV seronegative. The PBL were separated by density gradient centrifugation over Ficoll-Paque (Pharmacia, North Ryde, NSW, Australia), and washed three times in PBS. Cells were counted and the cell viability was> 95% by trypan blue exclusion. For CD3 activation, donor B PBL at 2 10⁶ cells/mL were incubated overnight at 37°C, 5% CO₂ in RPMI media containing 25 mmol/L HEPES (Gibco BRL, Melbourne, Australia), 2 mmol/L glutamine, 10% foetal bovine serum, 100 U penicillin, 100 g/mL streptomycin (Trace Biosciences, Castle Hill, NSW, Australia), and 100 ng/mL anti-CD3 antibody (Dako, NSW, Australia). Cells were washed in PBS and counted; cell viability remained> 95%. Mice were reconstituted with human PBL by intraperitoneal injection of PBL in 0.5 mL PBS. Control mice received PBS.

Measurement of human IgG in sera

Mice were bled periodically by retro-orbital eye bleed. The sera were collected after centrifugation of blood and HIgG levels were measured using an inhibition ELISA. Briefly, all ELISA plates were coated with 0.5 g/well HIgG and then blocked for 1 h at room temperature with PBS/1% BSA. Following washing of plates with PBS/0.05% Tween-20, standards of known concentration of HIgG or test sera from SCID mice were used to inhibit the binding of peroxidase-conjugated rabbit anti-human IgA + IgG + IgM (Dako, Botany, NSW, Australia); dilution 1:5000. o-Phenylenediamine reaction products were detected by tetramethylbenzidine (TMB) substrate (Dako) and absorbances at 450 nm were measured by an automated microplate reader.

Polymerase chain reaction detection of human HLA-DRb DNA in SCID periphery

Genomic DNA was prepared from mouse peripheral blood using a one-step guanidinium hydrochloride extraction procedure.¹⁵ The DNA was amplified by polymerase chain reaction (PCR) in a standard 50 L reaction containing 10 mmol/L Tris/HCl (pH 8.4), 50 mmol/L KCl, 1.5 mmol/L MgCl₂, 0.01% (w/v) gelatin, 0.2 mmol/L of each deoxynucleotidetriphosphate (dNTP), 25 pmol each of forward and reverse primer, and 1.25 units of Taq DNA polymerase (Promega, Sydney, Australia). The following oligonucleotide primers specific for human HLA-DR were used,⁸ (forward 5'-TTCTTCAATGGGACGGAGCG-3' and reverse 5'-GCCGCTGCACTGTGAAGCTCTC-3'). Polymerase chain reaction amplification was carried out for 1 min each at 95°C, 68°C, and 72°C for 40 cycles. Human genomic DNA was included as a positive PCR control; negative PCR controls contained all the reaction components for PCR except template DNA. The PCR products (5 L) were electrophoresed in a 1.25% agarose gel and the length of the products assessed by comparison with molecular size standards after staining with ethidium bromide. The specificity of each PCR product was confirmed by Southern blotting and hybridization with an internal HLA-DR-specific probe (5'-CTGGAACAGCCAGAAGGAC-3') that was end-labelled with [³²P]-ATP.

Immunohistochemistry

All surviving mice were killed 8 weeks after reconstitution. Mouse organs (spleen, lymph nodes, thymus, liver and lung) were taken and fixed in 10% formalin. Paraffin-embedded SCID tissue sections were stained with a mouse anti-human CD45 (leucocyte common antigen) monoclonal antibody. Tissue sections were deparaffinized, incubated for 5 min in 0.6% hydrogen peroxide/methanol to block endogenous peroxidase, and then incubated for 15 min at 95°C in 10 mmol/L sodium citrate (pH 6.0) target retrieval buffer. After washing for 5 min in TBS/1% BSA, endogenous avidin and biotin sites were blocked using a commercially available kit (Dako). Tissue sections were blocked for 20 min in 20% normal rabbit serum before incubation for 30 min at room temperature with antihuman CD45 primary antibody; dilution 1:50 (Dako). Tissue sections were washed in trisbuffered saline (TBS)/1% BSA and then incubated for 30 min with rabbit anti-mouse biotinylated secondary antibody; dilution 1:200 (Dako). Following washing in TBS/1% BSA, positive cells were detected using StreptABComplex/horseradish peroxidase (HRP) and diaminobenzidine (DAB) according to the manufacturer's instructions (Dako). The same protocol was used for staining of SCID tissues with an anti-human CD3 polyclonal antibody; dilution 1:50 (Dako), except that slides were incubated for 30 min in target retrieval buffer, blocked in 20% normal swine serum and incubated with an anti-rabbit biotinylated secondary antibody; dilution 1:300 (Dako). All antibodies and sera were diluted in TBS/1% BSA. Positive controls for tissue staining included human tonsil for human CD45 and CD3. Negative control staining of tissue sections was carried out using an irrelevant isotype-matched antibody, instead of primary antibody. All tissue sections were counterstained with Meyer's haematoxylin.

Results and discussion

Human IgG and HLA-DRb DNA in Hu-PBL-SCID periphery

Human IgG levels were measured in PBL-reconstituted SCID mice sera at several time-points following the transfer of human PBL (Table 1). Human IgG was detected at 1 week after reconstitution, albeit at relatively low levels, in animals that received at least 1 10⁷ PBL. The levels of HIgG continued to rise over a period of 8 weeks in all PBL-reconstituted SCID, and reached levels comparable to those detected in the original donor PBL; donor A, 2907 g/mL and donor B, 3160 g/mL. The long-term production of human antibodies in Hu-PBL-SCID has previously been demonstrated.^{4, 5, 6} Together, these findings indicate that the SCID environment supports all the necessary immune factors required for B cell function and antibody production. Very low levels of HIgG were detected in SCID A4, suggesting that more than 1 10⁷ cells may be required for efficient engraftment of human PBL into SCID mice. However, more animals would need to be examined to establish a correlation between engraftment and cell number. The number of cells to be transferred has previously been shown to be an important factor in determining the success of repopulation of human cells into SCID.³ Negligible levels of HIgG, or no HIgG, were detected in PBS control mice, A5 and B5, at all of the time points tested. Of the 10 mice examined, two died, one each at 6 and 8 weeks after reconstitution. These two SCID mice had relatively high HIgG levels in their sera when compared to the other animals in the group.

Table 1 - Human (H) IgG levels and HLA-DR DNA detection in the periphery of SCID mice reconstituted with human peripheral blood lymphocytes.

Full table (32K)

The HLA-DR human-specific DNA was detected in the majority of SCID mice periphery at 3 weeks post-reconstitution (Table 1; Figure 1). There were no HLA-DR PCR amplification signals detected in control SCID, or in SCID A4, at any time point during the analysis. The integrity of each genomic DNA sample was confirmed by PCR amplification employing primers specific for mouse whey acidic protein (not shown).

Figure 1.

Polymerase chain reaction (PCR) detection of human-specific HLA-DR DNA in the periphery of severe combined immunodeficiency (SCID) mice reconstituted with human PBL. Genomic DNA was prepared from mouse peripheral blood obtained 3 and 6 weeks post-reconstitution of SCID mice with unstimulated PBL (SCID A1-A4) and anti-CD3-stimulated PBL (SCID B1-B4). The SCID mice A5 and B5 were PBS controls. The PCR amplification of mouse genomic DNA was performed with primers specific for human HLA-DR and the PCR products were detected by Southern blot hybridization with an internal oligonucleotide probe. Human genomic DNA was used as a positive control (Pos) for PCR. Exposure of blots was 3 h, and 25 min for 3-week and 6-week post-reconstitution, respectively. In the 6-week post-reconstituted SCID mice, there was no blood sample obtained for A1, and SCID B2 died before sample collection.

Full figure and legend (28K)

Human CD45⁺ cells in Hu-PBL-SCID lymphoid tissues

All surviving mice were killed at 8 weeks post-reconstitution. Upon macroscopic examination of mouse organs we observed splenomegaly as characterized by an enlarged spleen in a few cases, whereas all other organs appeared normal. There was no evidence of tumour growth in any of the xenografted SCID. Animals showed no physical symptoms of graft-versus-host disease; that is, hunched back, ruffled fur, diarrhoea or emaciation. Postmortem examination of xenografted SCID showed no marked lymphocytic infiltration of the intestinal crypts in the few gut mucosa biopsies taken for analysis, although some scattered CD45⁺ cells were detected (not shown). Previous studies of human T cell engraftment in SCID mice have shown that splenomegaly is a prominent feature of these chimeric animals and results from extensive murine extramedullary splenic haematopoiesis.^{12, 13, 14} In addition, while a graft-versus-host reaction has been demonstrated in some studies in the Hu-PBL-SCID,^{3, 12, 16} this appears not to be a major health problem.

Immunohistochemical staining for human CD45 in our Hu-PBL-SCID revealed the presence of CD45⁺ cells in the liver and lung, and in particular there was extensive human CD45 cellular infiltration into the spleen, lymph nodes and thymus, which, surprisingly, was equally pronounced in mice that received fresh unstimulated PBL and those that received CD3-stimulated PBL (Table 2). In contrast, we observed that the level of CD45 cell staining was greater in the lungs of animals that were reconstituted with CD3-stimulated PBL because all of these mice showed intense staining of CD45⁺ cells throughout the bronchioles, alveoli and lung parenchyma (Table 2). There were no other apparent differences observed between those animals that received unmanipulated PBL and those that received CD3-stimulated PBL because all animals, except SCID A4, showed evidence of human CD45⁺ cell engraftment. These findings indicate that abrogation of host NK activity by anti-asialoGM-1, which has previously been shown to enhance the engraftment of human PBL into SCID,^{11, 12, 13, 14, 15, 16} is not necessary for the establishment of chimeric Hu-PBL-SCID. A representative example of staining of tissues from a Hu-PBL-SCID is given in Figure 2. In contrast, no human CD45⁺ cells were detected in the control mice. In the spleens of some SCID, we found that human CD45⁺ cells localized within the peri-arteriolar sheaths of the white pulp, whereas there was no specific localization of CD45⁺ cells observed within the lymph nodes and thymus. These findings support the migration specifically of human cells into Hu-PBL-SCID tissues. To rule out the possibility of cross-reactivity of the anti-human CD45 antibody with mouse leucocytes, we also carried out immunohistochemical staining of C57/BL6 mouse spleen with an anti-mouse and anti-human CD45 antibody. Numerous murine CD45⁺ cells were detected in the C57/BL6 spleen whereas staining for human CD45⁺ cells was negative (not shown).

Figure 2.

Immunohistochemical staining of human (Hu)-PBL-severe combined immunodeficient (SCID) mouse tissues for human CD45⁺ cells. Paraffin-embedded tissue sections of (a) spleen, (b) lymph nodes, (c) thymus, (d) liver and (e) lung were obtained from a SCID mouse (A3) 8 weeks following intraperitoneal injection of 2 10⁷ unstimulated PBL. Tissue sections were stained with anti-human CD45 and positive cells detected with diaminobenzidine (DAB) substrate (brown colour). Massive infiltration of human CD45⁺ cells were observed in the spleen, lymph nodes and thymus; numerous CD45⁺ cells were also detected in the liver whereas a few scattered CD45⁺ cells were observed in the lung. Note that in this case CD45⁺ cells localized within the peri-arteriolar sheaths in the spleen. Magnification 100; 250 in (b) and (c).

Full figure and legend (204K)

Table 2 - Immunohistochemical staining of human CD45 and CD3 in Hu-PBL-SCID tissues.

Full table (24K)

Human CD3⁺ T cells predominate in Hu-PBL-SCID lymphoid tissues

To determine the extent of CD3⁺ T cell infiltration in our SCID chimeras, we performed immunohistochemical staining of SCID tissues with an anti-human CD3 antibody. CD3⁺ T cells were detected in the liver and lung, and predominated in the spleen, lymph nodes, and thymus (Table 2). We found no major differences between animals that received unmanipulated PBL and those that received CD3-stimulated PBL regarding human CD3 T cell engraftment in lymphoid tissues. Interestingly, CD3⁺ T cell staining was observed in the thymus when animals received unstimulated PBL. These findings indicate that T cell migration from the peritoneal cavity and entry into the thymus can occur in SCID mice repopulated with unstimulated PBL. A representative example of staining of tissues from a Hu-PBL-SCID is given in Figure 3. T cell entry into the thymus of Hu-PBL-SCID has only previously been demonstrated following activation of PBL,¹² or treatment with growth hormone,¹⁴ indicating that different homing signals may be required for T cell entry into different lymphoid tissues.

Figure 3.

Immunohistochemical staining of human (Hu)-PBL-severe combined immunodeficient (SCID) mouse tissues for human CD3⁺ T cells. Paraffin-embedded tissue sections of (a) spleen, (b) lymph nodes, (c) thymus, (d) liver and (e) lung, were obtained from a SCID mouse (A3) 8 weeks following intraperitoneal injection of 2 10⁷ unstimulated PBL. Tissue sections were stained with anti-human CD3 and positive cells detected with diaminobenzidine (DAB) substrate (brown colour). Engraftment of human CD3 T cells was observed in the spleen, lymph nodes, thymus, and liver whereas fewer T cells were detected in the lung. Magnification 100; 250 in (b) and (c).

Full figure and legend (204K)

There have been many reports of the engraftment of SCID mice with human PBL; however, successful engraftment, or enhanced engraftment, has previously been achieved largely with some form of manipulation of the human PBL, irradiation of the host, or by removal of host NK cells.^{8, 11, 12, 13, 14} Unlike previous studies,¹² we found no difference in the engraftment of SCID mice by human PBL when using CD3-activated as opposed to unstimulated PBL as assessed by immunohistochemistry. Moreover, the detection of human specific HLA-DR DNA by PCR, together with measurement of serum HIgG, allows an assessment of the level of human PBL engraftment at 3 weeks post-reconstitution, and does not require the animals to be killed.

Our data showed CD45 and in particular T cell engraftment in mouse lymphoid tissues of Hu-PBL-SCID, irrespective of whether PBL had been activated prior to transfer or not. Furthermore, T cell engraftment in the SCID thymus occurred following the transfer of unstimulated PBL indicating that this model may be suitable in further functional studies relating to the human immune response.

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Acknowledgements

This work was carried out in the Department of Surgery Research Laboratories at the Princess Alexandra Hospital. We thank the Renal Research Trust Fund for their continued support, and the Mayne Bequest Fund (University of Queensland). We are grateful to Dr Michael Walsh (University of Queensland Medical School Pathology) for mouse tissue sectioning, and Ms Anne-Louise Bullock (Department of Medicine) for kindly providing the mouse whey acidic protein PCR primers.