Increased lymphocytic infiltration of intestinal tissues has been observed in patients infected with HIV-1 and in SIV-infected rhesus macaques. To determine whether HIV-1 and SIV infections influence the homing of human and nonhuman primate PBMC to intestinal tissues, we engrafted SCID mice with human or nonhuman primate PBMC and infected them with either cell-free or cell-associated HIV-1 or SIV. In mice that received both PBMC and virus, human or nonhuman primate CD2+ T cells were found in intestinal tissues, primarily in the intraepithelial lymphocyte compartment and lamina propria. Immunomagnetic sorting revealed that these cells were derived from the CD4+ population. Using gag-specific primers, PCR analysis of these tissues detected the presence of HIV-1 proviral DNA. However, in SCID mice that were engrafted with either human or nonhuman primate PBMC and no HIV-1 or SIV, CD2+ T cells were not detected in intestinal tissues. These results indicate that HIV-1 and SIV can modulate the migratory properties of human and nonhuman primate T cells in the SCID mouse model.

Mucosal sites are the most common portals of entry for HIV-1 (1). After HIV-1 infection is established, the integrity of intestinal tissues is compromised, as evidenced by clinical manifestations including malabsorption, increased opportunistic infections, alterations in the intestinal morphology, and increased cellular infiltration; furthermore, CD4:CD8 T cell ratios within intestinal tissues are altered (2, 3, 4, 5, 6). These symptoms are also characteristic of SIV infection in rhesus and pig-tailed macaques, which is a widely used model system that reproduces most features of HIV infections in humans (7, 8, 9). In macaques it has been demonstrated that as early as 1 wk after infection, infiltration of SIV-infected T cells and macrophages occurs throughout the gut-associated lymphoid tissues (8). These findings suggest that infiltration of T cells may contribute to the retrovirus-associated dysfunction of the mucosal immune system.

Animal models may be useful to determine the role of retroviral infection in the recruitment of T cells to intestinal tissues and for understanding the underlying mechanisms of the virus-induced pathology. The human peripheral blood lymphocyte-engrafted SCID (hu-PBL-SCID)4 mouse model has been used to study HIV-1 pathogenesis. SCID mice engrafted with HIV-1-infected human PBMC develop some pathogenic features associated with HIV-1 infection in humans, including rapid CD4+ T cell depletion (10, 11, 12, 13, 14). This model has proven effective in examining the in vivo effects of different strains of HIV-1 with varying cellular tropism and cytopathogenicity. In addition, mice have been successfully engrafted with human PBMC from HIV-infected patients (13), thus providing a possible surrogate system not only to examine HIV pathogenesis, but also to determine the effects of retroviral therapy.

The current study was designed to determine whether SCID mice engrafted with human or nonhuman primate PBMC could provide a means to study the effects of HIV-1 or SIV on the homing patterns of T cells to the intestine and other tissues. Immunohistochemical techniques were used to compare tissues from uninfected and infected mice and to determine the extent of T cell localization. Additional methods were used to determine whether virus or proviral DNA could be detected in tissues in which engrafted T cells had localized. We report that infection with HIV-1 or SIV can induce T cell infiltration into intestinal tissues of SCID mice engrafted with human and nonhuman primate PBMC.

Male and female C.B-17 (scid/scid) mice (6–8 wk old) were obtained from the University of Alabama Animal Resources Program and were maintained in cages fitted with microisolators. Cages, bedding, food, and water were autoclaved before use; no antibiotics were administered.

Human PBMC were isolated from Leukopaks obtained from the American Red Cross (Birmingham, AL) by centrifugation on a Ficoll-Hypaque density gradient (Sigma Chemical Co., St. Louis, MO). All donors were tested for HIV-1 seropositivity and were negative. The cells were washed and resuspended in RPMI 1640 supplemented with l-glutamine, 100 U/ml penicillin, 100 mg/μl streptomycin, and 10% FBS. Viability was determined by trypan blue dye exclusion. The cells were resuspended at 2 to 3 × 107 viable cells/ml in supplemented RPMI 1640. PBMC from normal pig-tailed macaques and chimpanzees were purified from heparanized blood samples and processed as described above.

T cell-enriched fractions were prepared from PBMC by rosetting with 2-aminoethylisothiuronium bromide-treated SRBC overnight at 4°C (15). The rosetted cells (E+) were separated from the nonrosetted (E) cells by density gradient centrifugation. The E cells were taken from the gradient interface, and the T cell-enriched fraction (E+ cells) was prepared by hypo-osmotic lysis of the SRBC (cell pellet fraction of the density gradient). T cell subsets were isolated by immunomagnetic bead separation, according to the protocol of the manufacturer (Dynal, Oslo, Norway). Briefly, both E and E+ PBMC (2 × 107 cells/ml) were shaken gently on ice for 30 min with the appropriate mAb specific for human CD4 or CD8 conjugated to magnetic beads (Table I) at a bead:target cell ratio of 2:1. The CD4+ or CD8+ cells were magnetically separated from the cells in the supernatant (depleted population). Both populations of cells were washed, and the magnetic beads were removed from the positively selected cells with DETACHaBEAD (Dynal). The cells were extensively washed again in complete medium. The population purity was determined after each step by FACS analysis using mAbs against CD4, CD8, and CD19 (Table I) surface Ags, as described below. The procedure was repeated until a highly enriched population (>95%) of CD4+ or CD8+ cells was obtained. CD4-depleted populations from both the E and E+ cells were also recovered for further studies.

Table I.

Summary of antibodies used to identify human and nonhuman primate cells in engrafted SCID micea

SpecificityAntibodyUseSource
Human CD4 Dynabead M-450 CD4 Magnetic purification of CD4+ T cells from PBMC Dynal, Oslo, Norway 
Human CD8 Dynabead M-450 CD8 Magnetic purification of CD8+ T cells from PBMC Dynal 
Human MHC class I HLA-A,-B,-C–FITC Detection of human cells in peritoneal wash of hu-PBL-SCID mice by FACS Olympus Immunochemicals, Lake Success, NY 
Human CD2 CD2–FITC Detection in paraffin sections of human and nonhuman primate CD2+ cells in engrafted SCID tissues Becton Dickinson, San Jose, CA 
Human CD3 CD3-FITC Detection of human T cells in peritoneal wash of hu-PBL-SCID mice by FACS Becton Dickinson 
Human CD4 CD4-FITC   
Human CD8 CD8-PE   
Human CD19 CD19-PE Detection of human B cells in peritoneal wash of hu-PBL-SCID mice by FACS Becton Dickinson 
Mouse Ig Goat anti-mouse immunoglobulin–TRITC monoclonal Enhancement of CD2+ staining; detection of endogenous Ig production in SCID tissues Southern Biotechnology Associates, Inc., Birmingham, AL 
Human CD45RO CD45RO (UCHL-1) unlabeled Detection in paraffin sections of CD45RO+ (memory) cells in SCID tissues containing CD2+ cells Becton Dickinson 
Human CD8 CD8 unlabeled Immunoperoxidase detection of CD8+ cells in SCID tissues containing CD2+ cells Dako Corp., Carpinteria, CA 
Human CD57 CD57 unlabeled Immunoperoxidase detection of CD57+ (NK) cells in SCID tissues containing CD2+ cells Novocastra Laboratories, Ltd., Newcastle upon Tyne, UK 
Human CD3 CD3 rabbit polyclonal unlabeled Immunoperoxidase detection of CD3+ human and nonhuman primate cells in SCID tissues containing CD2+ cells Dako Corp. 
SpecificityAntibodyUseSource
Human CD4 Dynabead M-450 CD4 Magnetic purification of CD4+ T cells from PBMC Dynal, Oslo, Norway 
Human CD8 Dynabead M-450 CD8 Magnetic purification of CD8+ T cells from PBMC Dynal 
Human MHC class I HLA-A,-B,-C–FITC Detection of human cells in peritoneal wash of hu-PBL-SCID mice by FACS Olympus Immunochemicals, Lake Success, NY 
Human CD2 CD2–FITC Detection in paraffin sections of human and nonhuman primate CD2+ cells in engrafted SCID tissues Becton Dickinson, San Jose, CA 
Human CD3 CD3-FITC Detection of human T cells in peritoneal wash of hu-PBL-SCID mice by FACS Becton Dickinson 
Human CD4 CD4-FITC   
Human CD8 CD8-PE   
Human CD19 CD19-PE Detection of human B cells in peritoneal wash of hu-PBL-SCID mice by FACS Becton Dickinson 
Mouse Ig Goat anti-mouse immunoglobulin–TRITC monoclonal Enhancement of CD2+ staining; detection of endogenous Ig production in SCID tissues Southern Biotechnology Associates, Inc., Birmingham, AL 
Human CD45RO CD45RO (UCHL-1) unlabeled Detection in paraffin sections of CD45RO+ (memory) cells in SCID tissues containing CD2+ cells Becton Dickinson 
Human CD8 CD8 unlabeled Immunoperoxidase detection of CD8+ cells in SCID tissues containing CD2+ cells Dako Corp., Carpinteria, CA 
Human CD57 CD57 unlabeled Immunoperoxidase detection of CD57+ (NK) cells in SCID tissues containing CD2+ cells Novocastra Laboratories, Ltd., Newcastle upon Tyne, UK 
Human CD3 CD3 rabbit polyclonal unlabeled Immunoperoxidase detection of CD3+ human and nonhuman primate cells in SCID tissues containing CD2+ cells Dako Corp. 
a

Unless otherwise noted, all reagents used were murine mAbs.

The MOLT 4 clone 8 (MOLT) T cell line was maintained in supplemented RPMI 1640. A viral stock was produced by infecting MOLT cells with HIV-1IIIB. Measurements of reverse transcriptase (RT) activity were used to determine HIV-1 infection (16); viral stocks were generated from the culture supernatants and titrated as described previously (16). The stock chosen for use in the hu-PBL-SCID mice was determined to have a 105 median tissue culture infectious dose (TCID50)/ml supernatant on the MOLT cell line.

Several HIV-1 strains were used to infect chimpanzee cells. Additionally, the SIVsmmPBj14 strain of SIV was used to infect macaque cells. Further characterization of this virus was detailed previously (17).

Influenza virus strain A/Udorn//307/72 (H3N2) (a gift from Dr. B. Murphy, National Institutes of Health, Bethesda, MD) was grown and isolated as previously described (18).

A total of 53 SCID mice were injected i.p. with 2 to 3 × 107 freshly isolated human PBMC (hu-PBL-SCID) or immunomagnetically sorted cell populations (Table II). In addition, three SCID mice that did not receive any human cells were used to obtain control samples for histology and FACS analysis. Four nonengrafted SCID mice were infected with HIV-1IIIB as additional control samples for histology and HIV-1 p24 gag detection. Two weeks after injection of human PBMC, some of the engrafted mice were infected i.p. with 105 TCID50 of HIVIIIB (Table II). Mice were killed 21 days after engraftment with human cells.

Table II.

Number of mice engrafted with human lymphocytes

Total PBMC (No. of Mice)CD4+a (No. of Mice)Purityb (%)CD8+a (No. of Mice)Purity (%)CD4-depletedad (No. of Mice)Puritye (%)
HIV-1 37 99 ND ND 97 
HIV-1+ 10 (1)ac 99 96 97 
Influenza+ ND ND ND ND ND ND 
Total PBMC (No. of Mice)CD4+a (No. of Mice)Purityb (%)CD8+a (No. of Mice)Purity (%)CD4-depletedad (No. of Mice)Puritye (%)
HIV-1 37 99 ND ND 97 
HIV-1+ 10 (1)ac 99 96 97 
Influenza+ ND ND ND ND ND ND 
a

SCID mice were engrafted with PBMC from same donor. ND, not determined.

b

Purity of the immunomagnetically sorted cell population, as determined by FACS staining.

c

One mouse received reconstituted PBMC in which the E+ population was added back to the E population after E-rosetting.

d

The CD4-depleted E+ population was added to the CD4-depleted E population.

e

Purity of CD4-depleted population, as determined by percent CD4+ contamination.

A total of 20 SCID mice were inoculated i.p. with 2 × 107 chimpanzee PBMC, and 2 to 3 wk later, with 2000 TCID50 of various HIV-1 strains. In addition, PBMC from pig-tailed macaques were stimulated with Con A and infected with SIVsmmPBj14 in vitro before i.p. inoculation of 2.5 to 5 × 107 cells into 30 mice. Three mice were inoculated with uninfected macaque PBMC. Characterization of the chimpanzee and pig-tailed macaque PBL-SCID models will be reported elsewhere (17).

Human PBMC were infected with the influenza virus at 5 to 10 multiplicity of infection for 1 h at 37°C in 1 ml of RPMI 1640 (18). The cells were washed extensively, and viability was determined as described above. Aliquots of 2.5 × 107 cells in 0.5 ml of supplemented RPMI 1640 were injected i.p. into SCID mice. A total of eight mice were engrafted with influenza-infected human PBMC (Table II).

Peritoneal cells from hu-PBL-SCID mice were recovered by flushing the cavity with 3 ml of PBS. Sections of the spleen and small intestine were collected for histology; in addition, mesentery, liver, kidney, large intestine, thymus, pleural fat, lungs, salivary gland, testes, ovaries, and fallopian tubes from some animals were collected for histologic examinations. Cells were isolated from the spleen by mechanical dissociation and analyzed by FACS. Intestinal tissues were obtained from SCID mice reconstituted with PBMC from chimpanzees (20 mice) and pig-tailed macaques (33 mice).

Peritoneal and splenic lymphocyte populations were analyzed by flow cytometry, using a mAb against human MHC class I (Table I) to distinguish the human cells from the resident mouse cells. In addition, peritoneal cells were stained with Abs specific for human CD3, CD8, and CD4 (Table I) to determine the percentage of each T cell subset.

Tissues were fixed in acid alcohol (95% ethanol and 5% glacial acetic acid), embedded in low melting point paraffin, and processed according to the method of Sainte-Marie (19). For analysis of the T cell populations in intestinal tissues of engrafted mice, immunofluorescence staining was performed using a primate cross-reactive mAb to CD2-FITC (Table I). To enhance detection of CD2 as well as to check for mouse Ig production (“leakiness”), a goat anti-mouse Ig conjugated to TRITC (Table I) was added as a second-step reagent. The tissue sections ranged in length from 12 to 14 mm and were scored for CD2+ cells per section as follows: +, 1 to 20 cells; ++, 21 to 50 cells; +++, 51 to 100 cells; and ++++, >101 cells. In some tissues that were positive for CD2, immunofluorescence staining was performed using an unlabeled mAb (Table I) known to react with CD45KO Ag in paraffin-embedded tissues followed by the goat anti-mouse Ig-TRITC conjugate. For immunoperoxidase staining of the hu-PBL-SCID paraffin-embedded sections, unlabeled mAbs against CD8 and CD57 (Table I) as well as an unlabeled polyclonal reagent against CD3 (Table I) were used with serial sections of intestinal tissues. Biotinylated secondary Abs were used in a standard ABC assay (Elite Vectastain ABC kit for mouse or rabbit, Vector Laboratories, Burlingame, CA), and color development was achieved with 3-amino-9-ethylcarbazole (Vector). All Abs were shown to recognize the appropriate lymphocyte populations in normal human tissues from tonsil, spleen, and intestine (Tissue Procurement Laboratory, University of Alabama, Birmingham, AL) embedded in paraffin. Additionally, the CD2 Ab reacted with T cell areas in paraffin-embedded spleen from rhesus macaques. To ensure that reagents did not detect mouse lymphocytes, sections of normal spleen and intestine from BALB/c mice were also tested with the anti-human Abs.

The recovered peritoneal cells (0.5–2 × 106 cells) from uninfected and HIV-1-infected hu-PBL-SCID mice were resuspended in 1 ml of complete RPMI 1640 supplemented with recombinant human IL-2 (0.5 ng/ml; R&D Systems, Minneapolis, MN). PHA-stimulated human PBMC were washed and resuspended at a concentration of 2 × 107 cells/ml in complete RPMI 1640 supplemented with recombinant human IL-2. Equal volumes of stimulated PBMC and recovered peritoneal cells were cocultured for 1 mo. The culture supernatants were monitored weekly for the presence of HIV-1 p24 capsid Ag using a commercial ELISA kit (Coulter Corp., Hialeah, FL). Cultures were considered positive if two consecutive weekly samples contained >200 pg/ml of p24 Gag Ag.

Genomic DNA was isolated from hu-PBL-SCID intestine and spleen using the QIAmp Tissue Kit (Qiagen, Inc., Chatsworth, CA). Three hundred nanograms of total DNA was added to 25 μl of a standard PCR reaction mixture that also contained 0.4 mM each of the HIV-1 gag gene-specific primer pair SK38 and SK39 (Research Genetics, Huntsville, AL). The amplified DNA was labeled with digoxigenin (DIG) by adding DIG-conjugated dUTP (Boehringer Mannheim, Indianapolis, IN) to the PCR mixture. A 115-bp DNA fragment of the gag gene was amplified (35 cycles) using standard PCR conditions. After separation on a 3% agarose gel and transfer to a nylon membrane, DIG-labeled PCR products were detected with anti-DIG-alkaline phosphatase conjugate and a chemiluminescent substrate, according to the instructions of the manufacturer (Boehringer Mannheim).

An average of 2.4 × 106 total cells (range, 0.5–6.5 × 106) was recovered from peritoneal lavages of hu-PBL-SCID mice. To determine whether the SCID mice were engrafted with human PBMC, cells were analyzed by two-color FACS analysis for human MHC class I Ags (Fig. 1,A). In addition, recovered peritoneal cells were stained with mAbs to human CD3, CD4, and CD8 (Fig. 1, B and C, and Table III) to determine the percentage of recovered T cells. At 2 wk after engraftment, but before HIV-1 inoculation, the normal CD4:CD8 ratio of 2:1 in human PBMC was inverted (1:2) in peritoneal cells that were recovered. At 7 days after HIV-1 inoculation, the ratio was 1:3 (Table III). Interestingly, the percentage of CD8+ cells in the recovered cell population also declined. Since a substantial number of CD8+ cells can often be recovered from the spleen (Fig. 1 C) and thymus of hu-PBL-SCID mice, movement of these cells into these organs may account for the decrease observed in the peritoneal cavity.

FIGURE 1.

Flow cytometric analysis of human cells recovered from peritoneal lavage (left panel) and spleen (right panel). Representative data from one mouse are shown. A, Cells were stained with mAb against human MHC class I (HLA-A, -B, and -C) to distinguish the human cell population. B, Human T and B cell populations were analyzed using anti-human CD3 and CD19 mAb. C, Percentages of T cell subsets were determined with mAb against human CD4 and CD8.

FIGURE 1.

Flow cytometric analysis of human cells recovered from peritoneal lavage (left panel) and spleen (right panel). Representative data from one mouse are shown. A, Cells were stained with mAb against human MHC class I (HLA-A, -B, and -C) to distinguish the human cell population. B, Human T and B cell populations were analyzed using anti-human CD3 and CD19 mAb. C, Percentages of T cell subsets were determined with mAb against human CD4 and CD8.

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Table III.

Phenotype of peritoneal cells recovered from uninfected and HIV-1-infected hu-PBL-SCID micea

PhenotypeUninfected (%)bInfected (%)
HLA-ABC+ 44 ND 
 (19–86)  
CD3+ 41 ND 
 (10–80)  
CD19+ ND 
 (0–6)  
CD4+ 13 <1 
 (11–15) (0.3–0.9) 
CD8+ 28 
 (15–68) (1–4) 
PhenotypeUninfected (%)bInfected (%)
HLA-ABC+ 44 ND 
 (19–86)  
CD3+ 41 ND 
 (10–80)  
CD19+ ND 
 (0–6)  
CD4+ 13 <1 
 (11–15) (0.3–0.9) 
CD8+ 28 
 (15–68) (1–4) 
a

An average of 2.4 × 106 cells was recovered by peritoneal lavage of SCID mice engrafted with human PBMC. ND, not determined.

b

Data are expressed as the mean percentage (range), as determined by FACS analysis.

Because detection of viral Ags by ELISA is more sensitive than that by classical RT assays, ELISA kits were used to detect HIV p24 Gag Ag in supernatants from cocultured peritoneal cells. Virus replication was demonstrated by detection of p24 Gag in 68% of the cocultures of peritoneal cells from mice engrafted with human PBMC and infected with cell-free HIV-1. On the average, over 300 pg/ml of viral p24 was detected in the cultures by the fourth week of coculture. In mice that received human cells but no virus, no detectable levels of p24 (<0.055 OD) were found in peritoneal cocultures.

In both uninfected and HIV-1-infected hu-PBL-SCID mice, T cells were found in the spleen and peritoneal lavage (Fig. 1, B and C). To reduce the biohazard associated with HIV-1-infected tissues, all samples were fixed and paraffin embedded; however, this process destroys some cell surface epitopes, including CD4. Thus, Abs against CD2 were used to detect human T cells in the tissue sections. In the uninfected engrafted SCID mice, T cells were mainly seen in the spleen and were occasionally detected in the thymus and mesentery, but not in intestinal tissues. Of interest, CD2+ T cells were found not only in the spleens of the HIV-1-infected hu-PBL-SCID mice, but also in the intestines (Fig. 2,B and Table IV). T cells were located in the lamina propria (LP), intraepithelial lymphocyte (IEL) compartment, and an occasional Peyer’s patch in the small intestine (Fig. 2,B and Table V). The number of CD2+ cells per section ranged from 10 to >100 (Table IV). T cells in the small intestine displayed an uneven distribution among the villi. Along the length of the small intestine, some areas contained large numbers of T cells within several villi, followed by areas where only a few T cells were detected. T cells were also detected in the large intestines. To analyze the phenotype of the CD2+ cells further, serial sections were reacted with anti-human CD8 and CD57 (to exclude the NK cell population); no positive cells were detected (Fig. 3, C and D, and Table VI). Polyclonal Abs against the cytoplasmic tail of human CD3 were also used to detect T cells (Fig. 3 E). In the majority of experiments, CD3+ cells correlated with areas containing CD2+ cells; however, this polyclonal reagent demonstrated some cross-reactivity to murine T cell epitopes. In two of the control mice that did not receive human cells, low numbers of CD3+ cells were detected (3–10/section); however, these mice were not “leaky,” as shown by both negative serum Ab levels and immunofluorescence staining of murine tissues for mouse Ig. In serial sections of intestinal tissues that were shown to contain CD2+ cells, a mAb against CD45RO was used to characterize these cells further. Although this Ab did identify CD45RO+ cells in SCID intestinal tissues, which correlated with the location of CD2+ cells, the surface fluorescence intensity was weak (data not shown). However, similar weak surface CD45RO+ fluorescence was observed using the same mAb on normal human tissues, such as small intestine and spleen.

FIGURE 2.

Immunofluorescence staining of small intestine taken from SCID mice engrafted with human (A and B) or macaque (C and D) PBMC and stained with anti-human CD2 mAb. A, Small intestine from an uninfected hu-PBL-SCID mouse. B, CD2+ cells in the LP and IEL of an HIV-1IIIB-infected hu-PBL-SCID mouse. C, CD2+ cells in a Peyer’s patch and (D) the LP and IEL of a SCID mouse engrafted with SIVsmmPBj14-infected macaque PBMC. CD2+ staining (FITC) of the tissue sections in A, C, and D was computer enhanced (magnification, ×100). Detection of CD2+ cells in B was enhanced using a TRITC-conjugated secondary Ab against mouse Ig. No staining by the secondary Ab alone was detected.

FIGURE 2.

Immunofluorescence staining of small intestine taken from SCID mice engrafted with human (A and B) or macaque (C and D) PBMC and stained with anti-human CD2 mAb. A, Small intestine from an uninfected hu-PBL-SCID mouse. B, CD2+ cells in the LP and IEL of an HIV-1IIIB-infected hu-PBL-SCID mouse. C, CD2+ cells in a Peyer’s patch and (D) the LP and IEL of a SCID mouse engrafted with SIVsmmPBj14-infected macaque PBMC. CD2+ staining (FITC) of the tissue sections in A, C, and D was computer enhanced (magnification, ×100). Detection of CD2+ cells in B was enhanced using a TRITC-conjugated secondary Ab against mouse Ig. No staining by the secondary Ab alone was detected.

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Table IV.

Summary of intestinal T cells in human and nonhuman primate PBMC-engrafted SCID micea

Human PBMCChimpanzee PBMCPig-Tailed Macaque PBMC
No. of Mice% of Miceb CD2+Meanc scoreNo. of Mice% of Mice CD2+Mean scoreNo. of Mice% of Mice CD2+Mean score
Infected (HIV-1/SIV) 10 62 ++ 20 75 30 76 +++ 
Uninfected 37 ND ND ND 
Infected (influenza) ND ND ND ND ND ND 
Human PBMCChimpanzee PBMCPig-Tailed Macaque PBMC
No. of Mice% of Miceb CD2+Meanc scoreNo. of Mice% of Mice CD2+Mean scoreNo. of Mice% of Mice CD2+Mean score
Infected (HIV-1/SIV) 10 62 ++ 20 75 30 76 +++ 
Uninfected 37 ND ND ND 
Infected (influenza) ND ND ND ND ND ND 
a

SCID mice engrafted with total PBMC from each primate source. ND, not determined.

b

Percentage of mice with CD2+ T cells in the small intestine, as determined by immunofluorescence staining.

c

At least two sections of small intestine from each mouse were scored. The score was per section for number of CD2+ cells present. +, 1 to 20 cells; ++, 21 to 50 cells; +++, 51 to 100 cells; and ++++, >101 cells.

Table V.

Summary of intestinal T cells within different intestinal sites (IEL, LP) in human and nonhuman primate PBMC-engrafted SCID micea

Species of PBMC DonorVirusNo. of MiceNo. SectionsbMean % in IELc (Range)Mean % in LPc (Range)
Human HIV-1 13 15 88 12 
    (67–100) (0–33) 
Chimpanzee HIV-1 13 18 82 18 
    (58–100) (0–42) 
Macaque SIV 10 97 
    (88–100) (0–12) 
Species of PBMC DonorVirusNo. of MiceNo. SectionsbMean % in IELc (Range)Mean % in LPc (Range)
Human HIV-1 13 15 88 12 
    (67–100) (0–33) 
Chimpanzee HIV-1 13 18 82 18 
    (58–100) (0–42) 
Macaque SIV 10 97 
    (88–100) (0–12) 
a

SCID mice engrafted with total PBMC from each primate source.

b

Paraffin sections from SCID intestinal tissues containing CD2+ primate cells were analyzed for CD3, as determined by immunoperoxidase staining.

c

The total numbers of CD3+ cells within the IEL and lamina propria were quantitated per section. The data are expressed as the mean percentage within each compartment as calculated from all sections.

FIGURE 3.

Immunoperoxidase staining of human tonsil sections stained with anti-human CD8 mAb (A) and anti-human CD57 mAb (B). Immunoperoxidase staining of small intestine sections from the same HIV-1-infected hu-PBL-SCID mouse (C–E), and an HIV-1-infected SCID mouse engrafted with purified human CD4+ cells (F). Sections from intestinal tissues bearing CD2+ human cells stained with anti-human CD8 mAb (C), anti-human CD57 mAb (D), and anti-human CD3 polyclonal Ab (E and F) are shown. After development, sections were counterstained and visualized using brightfield microscopy.

FIGURE 3.

Immunoperoxidase staining of human tonsil sections stained with anti-human CD8 mAb (A) and anti-human CD57 mAb (B). Immunoperoxidase staining of small intestine sections from the same HIV-1-infected hu-PBL-SCID mouse (C–E), and an HIV-1-infected SCID mouse engrafted with purified human CD4+ cells (F). Sections from intestinal tissues bearing CD2+ human cells stained with anti-human CD8 mAb (C), anti-human CD57 mAb (D), and anti-human CD3 polyclonal Ab (E and F) are shown. After development, sections were counterstained and visualized using brightfield microscopy.

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Table VI.

Intestinal tissues from hu-PBL-SCID mice are negative for CD8 and CD57

PhenotypeaNo. of MiceNo. of SectionsMean Scoreb
CD8+ 
CD57+ 
PhenotypeaNo. of MiceNo. of SectionsMean Scoreb
CD8+ 
CD57+ 
a

Paraffin sections from hu-PBL-SCID intestinal tissues containing CD2+ cells were analyzed for CD8 and CD57, as determined by immunoperoxidase staining.

b

The same scoring scale used to quantitate CD2+ cells (Table IV) was applied. The score reflects the mean number of positive cells/section.

To determine whether another RNA virus which primarily infects via mucosal routes would also induce T cell migration into intestinal tissues, we engrafted SCID mice with influenza-infected human PBMC. As with other hu-PBL-SCID mice, T cells were detected in the spleen and peritoneal cavity. Measurable levels of human Ig were detected in the sera of these animals (data not shown). In five paraffin sections of intestine from three SCID mice engrafted with influenza-infected human PBMC, no CD2+ T cells were observed (Table IV).

Intestinal tissues collected from SCID mice engrafted with HIV-1-infected chimpanzee or SIV-infected pig-tailed macaque PBMC were also evaluated using the anti-human CD2 Ab. CD2+ cells were found in the intestinal tissues from SCID mice engrafted with SIV-infected macaque lymphocytes (Fig. 2, C and D, and Table IV) and were consistently detected in the mice given cell-associated virus. The number of CD2+ cells detected in the pig-tailed macaque PBMC-engrafted SCID mice ranged from 25 to >300/section of intestinal tissue, which was generally higher than those seen in the hu-PBL-SCID mice (Table IV). In mice that received uninfected macaque lymphocytes, no CD2+ cells were detected 21 days postengraftment. CD2+ cells were also observed in intestinal tissues from SCID mice that were engrafted with chimpanzee lymphocytes and subsequently infected with HIV-1. The number of CD2+ T cells in these latter mice ranged from 3 to 45 cells/section, which was considerably lower than that in hu-PBL-SCID mice (Table IV). Also, the distribution of CD2+ T cells in the intestines of these mice was similar to that observed in the hu-PBL-SCID mice, with localization to the IEL compartment, LP, and an occasional Peyer’s patch (Fig. 2, C and D, and Table V).

Because CD4 epitopes are not well preserved in paraffin sections, immunomagnetically sorted populations from the same human donor were used to determine the phenotype of the CD2+ T cells observed in the SCID mice. Purity was determined by FACS analysis before reconstituting SCID mice with either CD4+- or CD8+-purified populations (Table II). To ensure that E rosetting did not influence the engraftment, PBMC were E rosetted, and the E and E+ cells were pooled to form a reconstituted total PBMC population. To determine whether the intestinal CD2+ cells were from a cell population other than CD4+ cells, CD4+-depleted populations from both the E+ and E cells were pooled and engrafted into SCID mice. CD2+ cells were only detected in the intestines of HIV-1-infected mice engrafted with total reconstituted PBMC or CD4+-purified populations (Fig. 3,F and Table VII). These experiments indicate that the CD2+ cells in the intestine were probably derived from the CD4+ T cell population.

Table VII.

Summary of intestinal T cells in SCID mice engrafted with purified T cell subsets

Purified PopulationaNo. of MiceNo. of SectionsbMean Scorec
CD4+ ++ 
CD8+ 
CD4 depleted (CD8+
Purified PopulationaNo. of MiceNo. of SectionsbMean Scorec
CD4+ ++ 
CD8+ 
CD4 depleted (CD8+
a

SCID mice were engrafted with purified T cell subsets from the same PBMC donor.

b

Paraffin sections from SCID mice engrafted with purified T cell subsets were analyzed for CD3, as determined by immunoperoxidase staining.

c

The same scoring scale used to quantitate CD2+ cells (Table IV) was applied. The score shown reflects the mean number of positive cells/section.

Although some effort was made to identify p24 protein in the intestines of hu-PBL-SCID mice that contained CD2+ cells, fluorescence microscopy and RT-PCR yielded no positive results for p24 protein or mRNA (data not shown). However, spleen and intestinal tissues from the HIV-1-infected, human PBMC-engrafted SCID mice contained proviral copies of HIV-1, as determined by PCR amplification of a portion of the gag gene (Fig. 4, A and C). Similar sections from mice that were engrafted with uninfected human cells or from unengrafted SCID mice inoculated with HIV-1 were negative for HIV-1 proviral DNA (Fig. 4,B). DNA isolated from uninfected and infected U937 cells, a promonocytic cell line was used as negative and positive controls, respectively (Fig. 4 B). The detection of HIV-1 proviral DNA from various sections of the small intestine appeared to correspond with the distribution of CD2+ cells along the length of the small intestine.

FIGURE 4.

Detection of HIV-1 proviral DNA in tissues from hu-PBL-SCID mice. After PCR amplification, the DIG-labeled DNA products were detected by anti-DIG-alkaline phosphatase and reacted with a chemiluminescent substrate. A, HIV-1 gag DNA was detected in splenic tissue isolated from three (labeled 1, 2, and 3) hu-PBL-SCID mice infected with HIV-1. Multiple samples of splenic tissue from each mouse were processed (a–c). A promonocytic cell line, U937, either uninfected or infected with HIV was used as negative (−) and (+) controls, respectively, for all samples. B, Control DNA samples from SCID mice that were not engrafted with human PBMC, but were inoculated with HIV-1 (HIV+/PBMC); hu-PBL-SCID mice that were infected with HIV-1 (HIV+/PBMC+); hu-PBL-SCID mice that were not infected with HIV-1 (HIV-/PBMC+); U937 cell line infected or not infected with HIV-1. C, HIV-1 gag DNA detection in consecutive sections from along the small intestine (a–e: upper duodenum to lower ileum) from the same mice as those in A.

FIGURE 4.

Detection of HIV-1 proviral DNA in tissues from hu-PBL-SCID mice. After PCR amplification, the DIG-labeled DNA products were detected by anti-DIG-alkaline phosphatase and reacted with a chemiluminescent substrate. A, HIV-1 gag DNA was detected in splenic tissue isolated from three (labeled 1, 2, and 3) hu-PBL-SCID mice infected with HIV-1. Multiple samples of splenic tissue from each mouse were processed (a–c). A promonocytic cell line, U937, either uninfected or infected with HIV was used as negative (−) and (+) controls, respectively, for all samples. B, Control DNA samples from SCID mice that were not engrafted with human PBMC, but were inoculated with HIV-1 (HIV+/PBMC); hu-PBL-SCID mice that were infected with HIV-1 (HIV+/PBMC+); hu-PBL-SCID mice that were not infected with HIV-1 (HIV-/PBMC+); U937 cell line infected or not infected with HIV-1. C, HIV-1 gag DNA detection in consecutive sections from along the small intestine (a–e: upper duodenum to lower ileum) from the same mice as those in A.

Close modal

We report here that cell-free HIV-1 infection of SCID mice engrafted with human or chimpanzee PBMC or with SIV-infected pig-tailed macaque PBMC results in migration of CD2+ T cells from the peritoneal cavity into intestinal tissues. This phenomenon was not observed in the absence of lentiviral infection. HIV-1 and SIV are both members of the Retroviridae family of ssRNA viruses. In contrast, infection of hu-PBL-SCID mice with virus from another family of ssRNA viruses, influenza virus type A/Udorn, did not induce migration of T cells to intestinal tissues. The results suggest, therefore, that HIV-1 and SIV alter T cell migration in the engrafted SCID mouse. The finding that lymphocytes from different species can home to murine intestinal tissues is not entirely unexpected. Previous studies demonstrated that interspecies migration of lymphocytes into the LP of the small intestine can occur (20, 21). In fact, the homing receptors involved in this process share antigenic and structural features across species, suggesting a high conservation of lymphocyte-endothelial recognition systems during evolution (22).

Because it has been reported that HIV-1 infection of hu-PBL-SCID mice leads to a loss of CD4+ lymphocytes (10, 13, 14, 23), we determined CD4:CD8 ratios in cells recovered after peritoneal lavage and observed that a change occurred before HIV-1 infection. Another report of diminished CD4:CD8 ratios in the absence of HIV infection proposed that this decrease may result from the proliferation of CD8+ cells in response to murine Ags (23). After infection with HIV-1, the ratio was further decreased, perhaps as a result of migration of CD4+ T cells to the intestine.

For several reasons, we examined CD2 expression to identify primate T cell populations in tissues from engrafted SCID mice. First, we showed that the mAb to CD2 reacted with primate, but not murine, T cells. Second, the CD2 Ab recognized Ag in paraffin-embedded tissues. Finally, CD2 has been implicated as an accessory molecule in cell adhesion and cellular interactions and is highly expressed on the majority of normal human and murine intestinal T cells (24). CD2 can also be expressed on a subset of NK cells. However, consecutive sections tested for CD57, an NK cell Ag that is not destroyed by paraffin-embedding procedures, yielded negative results in intestinal tissues from engrafted SCID mice. This Ab was shown to detect NK cells in control paraffin-embedded human tissues, including intestine.

In all three primate PBMC-engrafted models, CD2+ cells were detected in the LP and IEL compartment of SCID intestines after HIV-1 or SIV infection. The results of engraftments with purified cell populations suggested that it was CD4+ cells that homed to the intestine after HIV-1 infection. In both the histology and the cell-sorting studies, no CD8+ cells were found in the intestinal tissues, nor were any NK cells detected. While it may not be surprising that HIV-1 induced only CD4+ T cells to migrate, the numbers of CD4+ cells found in the IEL compartment were unexpected, since the majority of murine and human IEL are CD8+; however, CD4+ cells can be detected in human and macaque IEL compartments (25, 26). Although we were unable to use immunofluorescence to determine whether the CD2+ cells in the paraffin-embedded tissues were CD4+, it is important to note that the human IEL compartment can also have cells with a CD4CD8 (double negative, 20–40%) or a CD4+CD8+ (double positive, 1–8%) phenotype (27). Similar phenotypes have also been observed within the murine IEL compartment (28). Negative CD8 staining suggests that these CD2+ cells are not double positive. Although our studies with enriched T cell populations demonstrated that the CD2+ cells in the SCID intestine can be derived from CD4+ cells, the possibility remains that these cells may have lost surface expression of CD4.

Since only SCID mice engrafted with primate PBMC and infected with HIV-1 or SIV harbored T cells in intestinal tissues, we suggest that these lentiviruses may alter the homing patterns of primate T cells in engrafted SCID mice and perhaps in infected humans. The nature of viral replication and infectivity may influence the number of cells homing to the intestinal tissues. The use of different primate species infected with viral strains that have diverse clinical consequences may provide insight into the mechanisms that are the basis of this migration. For example, SIVsmmPBj14 is the most virulent SIV strain, inducing acute disease and death within 1 to 2 wk in pig-tailed macaques (9). HIV-infected patients live several years before progressing to AIDS (29). On the other hand, chimpanzees infected with HIV-1 can develop some symptoms characteristic of human HIV-1 infections but, in general, do not progress to AIDS (30, 31). Lack of progression to disease may be linked to less efficient replication of HIV-1 by chimpanzee PBMC compared with that by human lymphocytes. This may explain why migration of CD2+ cells into the intestine of engrafted SCID mice from all three primates occurs, yet the numbers of CD2+ cells located in the intestines differ greatly. Detection of HIV-1 proviral DNA in intestinal tissues indicated that some of the migrating cells were infected. It is also possible that the number of lymphocytes in the intestinal tissues is correlated directly with the total number of infected cells in individual mice and/or the number of engrafted cells present at the time the mice were killed. It should also be noted that the macaque PBMC were infected in vitro before inoculation into SCID mice, whereas cell-free HIV-1 was injected 2 to 3 wk after engraftment with human or chimpanzee PBMC.

We have demonstrated that HIV-1 can apparently alter the primate T cell migration patterns in the chimeric SCID model. Taken together with the recent findings that chemokine receptors, such as CXCR4 (32, 33, 34, 35) and CCR5 (36, 37), serve as coreceptors for HIV-1 and that chemokines can cause migration of cells in vivo (38), we are currently investigating the effects of HIV-1 on the expression of mucosal homing receptors. The results from the present and planned studies may explain the increased cellular infiltration in mucosal tissues of asymptomatic HIV-infected patients as well as the early lymphocytic infiltration into intestinal tissues of SIV-infected macaques.

Since intraepithelial T cells appear to play a critical role in surveillance and repair of damaged intestinal epithelium (39), it is reasonable to speculate that these cells may participate in a carefully regulated process that maintains the integrity of the epithelium in the healthy intestine. Our data suggest that in vivo HIV-1/SIV infection may induce infiltration of T cells into the intestine, specifically the IEL compartment. Such infiltration may produce significant changes in the regulatory role played by the resident IELs, perhaps altering normal gut physiology. If such changes compromise the integrity of the mucosal barrier, the intestine might become more susceptible to those secondary infections common in HIV-infected individuals.

We thank Annette Pitts, Pam May, Jason Bunn, and John Campbell for their technical assistance. We acknowledge the support of Marion Spell and the CFAR FACS core facility. Finally, we thank Dr. Kevin McCarthy in the University of Alabama Digital Imaging Facility for his assistance with the computer imaging.

1

This work was supported by Grants AI07051, AI23952, AI28147, AI32377, and DE12146.

4

Abbreviations used in this paper: hu-PBL-SCID, human peripheral blood lymphocyte severe combined immunodeficient; RT, reverse transcriptase; TCID, tissue culture infectious dose; TRITC, tetramethylrhodamine isothiocyanate; DIG, digoxigenin; LP, lamina propria; IEL, intraepithelial lymphocytes.

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