Adoptive Transfer of Immunity with Intraepithelial Lymphocytes in Cryptosporidium parvum-Infected Severe Combined Immunodeficient Mice

Adoptive Transfer of Immunity with Intraepithelial Lymphocytes in Cryptosporidium parvum-Infected Severe Combined Immunodeficient Mice ANDREW A. ADJEI, PHD; ANAK K. SHRESTHA, MPH; MARA CASTRO, BSC; F. JAVIER ENRIQUEZ, MD, PHD

ABSTRACT: Background: Intestinal infections with the protozoan parasite Cryptosporidium parvum are prevalent in both immunocompetent and immunocompromised hosts. Although C parvum is an important cause of outbreaks and opportunistic infections worldwide, little is known about protective mucosal immune responses. This is in part because animal models of infection are limited to those with genetic or induced immunodeficiencies. Method: In this report, we isolated immune (primed) or nonimmune (unprimed) intraepithelial lymphocytes (IEL) from BALB/cJ mouse intestines, adoptively transferred them into C parvum-infected severe combined immunodeficient (SCID) mice, and evaluated infection and cell phenotype responses. Results: Control SCID mice that received no IEL shed large numbers of oocysts throughout the experimental period (day 18 to day 72). Transfer of primed IEL significantly reduced fecal oocyst shedding in recipient SCID mice

compared with SCID mice that received unprimed IEL or no IEL. SCID mice transferred with unprimed IEL shed variable numbers of fecal oocysts that increased and decreased in bursts until day 57 after infection. SCID mice transferred with primed IEL exhibited significantly higher proportions of T-cell receptor (TCR) ␣␤⫹, CD8⫹, and CD8␣␤⫹ EL compared with inoculated SCID mice that received unprimed or no IEL. Conclusion: We conclude that primed IEL from immunocompetent mice may influence protective mucosal response against cryptosporidiosis when transferred into SCID mice. In addition, the increased percentage of TCR ␣␤⫹, CD8⫹, CD8␣␤⫹ IEL in recipient SCID mice may reflect mucosal cell populations involved in these responses during chronic C parvum infection. KEY INDEXING TERMS: Intraepithelial lymphocytes; Cryptosporidium parvum; SCID mice. [Am J Med Sci 2000;320(5):304–309.]

C

CD4⫹, and both CD4⫹/CD8⫹ T cells are essential for development of protective immunity.3,4 Because C parvum invades the intestinal epithelium and is mainly confined to the villus and crypt epithelia during acute infections, it is reasonable to assume that intestinal mucosal responses may be involved in parasite clearance and merit examination.5 Unfortunately, a limiting factor for studying mucosal responses during cryptosporidiosis is the lack of small, immunocompetent animal models of infection. Mucosal immune response studies have been conducted frequently in immunodeficient mouse models, such as LP-BM5 retrovirus-infected, athymic nude, severe combined immunodeficient (SCID), several knock-out, and dexamethasone-treated mice.6 –12 Little information is available on protective mucosal immune responses during cryptosporidiosis. Recently, McDonald et al13 and Culshaw et al14 demonstrated the role of gut intraepithelial lymphocytes (IEL) in the development of resistance to C muris. Unfortunately, there are virtually no data

ryptosporidia are coccidian parasites that infect a wide variety of host species, including humans. One species, Cryptosporidium parvum, invades the intestinal epithelial cells of humans and other mammals, causing gastrointestinal illness.1 In immunocompetent persons, C parvum generally causes a self-limiting diarrheal illness. However, in immunocompromised hosts, such as AIDS patients, C parvum may cause a persistent cholera-like diarrheal disease that is sometimes life-threatening.2 Evidence from murine and human C parvum infections indicates that T-cell receptor (TCR) ␣␤⫹,

From the Department of Veterinary Science and Microbiology, University of Arizona, Tucson, Arizona. Submitted April 4, 2000; accepted June 2, 2000. This work was supported by National Institutes of Health grant AI39203. Correspondence: Andrew Anthony Adjei, Ph.D., Dept. of Vet. Science & Microbiology, Bldg. #90, Room 202, University of Arizona, Tucson, AZ 85721 (E-mail: [email protected]).

304

November 2000 Volume 320 Number 5

Adjei et al

demonstrating the contribution of mucosal immune responses to C parvum infection in immunodeficient mice. Because mucosal immunity, particularly IEL, against C parvum infection seems to operate in bovine models,15,16 we hypothesized that IEL could have a role in murine models. IEL constitute a unique lymphoid compartment of the intestinal mucosal immune system and may be considered the first line of defense against parasite infection.17 Most IEL express the CD8⫹ phenotype that can be either CD8 heterodimeric ␣␤-chains or homodimeric ␣␣-chains. Of the CD8 ␣␣ population, about 40% are TCR ␥␦ and 20% are TCR ␣␤.18,19 IEL provide a number of important immunological functions, including cytotoxicity and production of cytokines, including interferon-␥, interleukin (IL)-2, IL-4, IL-5, and tumor growth factor-␤.20 –22 Here, we demonstrate that immunity to C parvum infection in SCID mice was adoptively transferred with IEL from previously infected BALB/cJ mice. Materials and Methods Animals. Thirty-six 8-week-old female C.B-17 (H-2d) SCID mice (25–26 g) were obtained from Taconic Laboratories (Germantown, NY) and maintained at the University of Arizona Animal Care Facility. Mice were housed in microisolator cages in high-efficiency particulate air (HEPA)-filtered laminar flow racks and supplied with autoclaved water and sterilized normal mouse diet (Tekland; Harlan, Madison, WI) ad libitum. Eight-week-old female BALB/cJ (n ⫽ 12, 24 –26 g) mice used as the donor mice were obtained from Jackson Laboratories (Bar Harbor, ME). Parasites. Purified C parvum oocysts (IOWA isolate) were obtained from Pleasant Hill Farms (Mt. Pleasant, ID) and stored as previously described.23 Before mice were inoculated, oocyst preparations were treated with 1.75% (w/v) sodium hypochlorite (1 min, 22–23°C) and then washed with 0.025M phosphate buffred saline (PBS), pH 7.2. Experimental Design. Adult weight-matched SCID mice were divided randomly into 3 groups of mice. One group of mice (n ⫽ 12) received IEL from previously C parvum-primed BALB/cJ mice. The second group of SCID mice (n ⫽ 12) received IEL from unprimed BALB/cJ mice and served as transferred control. The third group of SCID mice (n ⫽ 12) received sterile PBS, and served as control. Primed donor BALB/cJ mice were inoculated intragastrically on 2 consecutive days with 106 C parvum oocysts in 100 ␮l of PBS using an 18-gauge gavage needle (Thomas Scientific, Swedesboro, NJ). Unprimed BALB/cJ mice received 100 ␮l of PBS per os. Fecal oocyst shedding in each inoculated BALB/cJ mice was determined twice a week for 60 days by immunofluorescence assay (IFA) as described previously.23 On day 60 postinoculation (PI), IEL were obtained from the small intestine of primed and unprimed BALB/cJ mice as described previously.24 –26 Day 60 PI for isolation of IELs was chosen based on a previous report that BALB/cJ mice recovering from C parvum infection exhibited no patent infection and that memory IEL were long lived.9 IELs were immunophenotyped by fluorescenceactivated cell-sorting analysis (FACS) and served as donor cells to recipient SCID mice. SCID mice were injected intraperitoneally with 106 purified IEL from the respective donor BALB/cJ mice. Four days after IEL adoptive transfer, all SCID mice groups were inoculated intragastrically with 106 C parvum oocysts. The 4-day waiting period after adoptive transfer was to allow IEL homing. Fecal pellets from each SCID mouse were collected twice weekly from days 3 to 72 PI to monitor oocyst shedding by IFA. Seventytwo days PI, SCID mice were killed by cervical dislocation after sedation, and small intestinal IELs were immunophenotyped by

THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

FACS. The experiment was repeated 3 times and all assays yielded consistently reproducible results. Preparation of Intraepithelial Suspension. Small intestines of donor BALB/cJ mice and recipient SCID mice were isolated as described previously by other investigators.24 –25 Briefly, small intestines were removed, from the pylorus to terminal ileum. Peyer’s patches, mesentery, and adherent tissue were carefully dissected macroscopically and fecal material was flushed from the lumen of each intestine using cold calciummagnesium-free Hanks’ balanced salt solution (Sigma Chemical Co., St. Louis, MO). The IEL were released by incubation in HBSS containing 10% fetal bovine serum, 1 mmol/L dithiothreitol, 1 mmol/L EDTA disodium, and 100 U/mL penicillin-streptomycin (all from Sigma) with continuous stirring for 6 20-min periods at 37°C. The IEL were pooled; filtered through nylon wool columns to remove dead cells, adherent cells, and tissue debris; and the cell suspensions centrifuged in Lympholyte-M (Cedarlane, Westbury, NY). Approximately 3 to 5 ⫻ 106 lymphocytes/ mouse were obtained from each density gradient separation. This procedure allows the isolation of IEL free from contamination by lamina propria lymphocytes.25,26 The viability of IELs from each donor was ⬎ 90% by trypan blue exclusion test. FACS Analysis. IEL suspensions (5 ⫻ 105 to 1 ⫻ 106 cells/mL) were washed separately with sterile PBS containing 0.01% sodium azide and 2% FBS. The cells were stained with fluorescein isothiocyanate-conjugated anti-CD4 monoclonal antibody (mAb; L3T4, Gk 1.5), anti-␥␦ TCR mAb (UC7–13D5), anti-CD8 mAb (Ly-3.2, 53–5.8), anti-CD8␤ mAb (H35–17.2), and phycoerythrinconjugated anti-CD3⑀ mAb (145–2C11), anti-␣␤ TCR mAb (H57– 597), anti-CD4 mAb (L3T4, Gk 1.5), anti-CD8 mAb (Ly-3.2, 53– 5.8), anti-CD8␣ 53– 6.7, or fluorescein isothiocyanate IgA (all from PharMingen, San Diego, CA). IgG/IgM (Kirkegaard and Perry Laboratories, Gaithersburg, MD) and flow cytometric analyses were performed as described previously.19,27 Histology. After obtaining IEL suspensions, intestinal segments were fixed in formal saline, embedded in paraffin wax. Sections (5 ␮m thick) were prepared and then stained with hematoxylin and eosin. Quantification of Oocysts in Feces. Fecal pellets from each inoculated BALB/cJ donor and recipient SCID mouse in each group were collected twice weekly from day 3 to days 60 and 72 PI, respectively, to score oocyst shedding by IFA as described previously.23 Statistical Analysis. Values for individual mice in each strain were averaged and significant differences among the means were analyzed by Student’s t-test and ␹2 test. P values ⬍ 0.05 were considered significant.

Results Donor IEL Priming. To determine whether IEL isolated were contaminated with lamina propria lymphocytes, intestinal segments were examined histologically. The results indicated that the villous architecture and basal membrane remained intact (data not shown). Flow cytometry was also employed to determine the major lymphocyte subpopulations present in the isolated IEL. Reports indicate that typical murine IEL from unprimed BALB/cJ donor mice consist predominantly of CD8⫹ and few Ig⫹ cells.13,28 In the present study, the percentage of IEL cell surface phenotypes in naive BALB/cJ mice were 85% CD8⫹, 23% CD4⫹, 75% ␣␤⫹, and ⱕ1% Ig⫹ cells as assessed by FACS. These results indicated that the IEL isolated contained little or no contamination with cells from the lamina propria. Similar results were observed in IEL surface phenotypes from primed BALB/cJ donor mice 60 days PI. 305

Adoptive Transfer of Immunity

Figure 1. Kinetics of fecal (pooled) oocyst shedding in C parvum-inoculated severe combined immunodeficiency mice after transfer with primed or unprimed IEL as assessed by immunofluorescence assay. *Values are significantly different from non-IEL-transferred control mice (P ⬍ 0.05).

BALB/cJ mice used as primed IEL donors were inoculated with 1 ⫻ 106 C parvum oocysts on 2 consecutive days and IEL were isolated 60 days later. As anticipated in these immunocompetent mice, the number of fecal oocysts after inoculation was transient from days 15 to 24 PI and then declined progressively until no oocysts were detected from days 33 to 60 PI (data not shown). Primed IEL isolated from these donor mice on day 60 PI and unprimed IEL from uninoculated donor mice were then adoptively transferred to SCID mice. Transfer of Primed IEL Confer Protection to C parvum Infection in SCID Mice. From day 18 to day 72 PI, high levels of oocyst shedding was noted in C parvum-inoculated SCID mice serving as control animals (no IEL transfer). The intensity of oocyst shedding in no-IEL-transferred SCID mice increased at days 27, 36, 51, 54, 57, 63, 66, 69, and 72 PI and was significantly higher than SCID mice transferred with either primed or unprimed IEL (P ⬍ 0.05, Figure 1). Inoculated SCID mice transferred with unprimed IEL shed a varying number of fecal oocysts throughout the experimental period (Figure 1), and on days 306

15 and 18 PI, that number was higher than that of SCID mice that received primed or no IEL (P ⬍ 0.05). Thereafter, the intensity of oocyst shedding in these unprimed IEL-transferred SCID mice decreased, with smaller peaks on days 36, 42, 48, 51, and 57 PI that were significantly higher than SCID mice transferred with primed IEL (P ⬍ 0.05). Sporadic fecal oocyst shedding (⬍ 5 oocysts per milligram of feces) was observed in inoculated SCID mice transferred with primed IEL from days 3 to 54 PI, and no oocysts were detected thereafter (Figure 1). The total mean number of oocysts per milligram of feces for all experimental days studied in inoculated SCID transferred with primed IEL (2.70 ⫾ 2.35) was lower compared with both inoculated SCID mice that received unprimed (10.65 ⫾ 12.65) or control no IEL-transferred mice (16.35 ⫾ 14.14), but without significant difference (P ⬎ 0.05). IEL Numbers. Seventy-two days PI, both inoculated SCID mice transferred with unprimed (17.11 ⫾ 1.55 ⫻ 106) or primed (18.11 ⫾ 5.72 ⫻ 106) IEL exhibited a significant increase in IEL numbers compared with inoculated SCID mice that received no (0.70 ⫾ 0.02 ⫻ 106; control) IEL (P ⬍ 0.01, November 2000 Volume 320 Number 5

Adjei et al

Table 1. Cell Surface Phenotype Analysis of IEL from C parvum-Inoculated SCID Mice after IEL Transfer Percent Expression of Cell Surface Markers ⫹

No IEL Transfer Unprimed IEL Transfer Primed IEL Transfer

⫹

TCR-␣␤

TCR-␥␦

13.61 25.53 47.10*

1.43 1.32 ⬍1

CD4⫹

CD8⫹

CD4␣␤⫹

CD4␥␦⫹

CD8␣␤⫹

CD8␥␦⫹

3.67 3.78 5.24

12.25 20.85 43.25*

2.38 3.53 5.81

⬍1 ⬍1 ⬍1

5.71 18.72 44.69*

⬍1 ⬍1 ⬍1

Mean values from 2 replicates. * Significantly different from both inoculated No IEL Transferred and Unprimed IEL Transferred SCID mice, P ⬍0.01.

P ⬍ 0.01, respectively). Inoculated SCID mice transferred with primed (18.11 ⫾ 5.72 ⫻ 106) or unprimed (17.11 ⫾ 1.55 ⫻ 106) IEL did not differ significantly in IEL cell numbers (P ⬎ 0.05). The average number of total IEL in naive (uninoculated) SCID mice was 0.89 ⫾ 0.26 ⫻ 106. IEL Phenotypes. Analysis of IEL surface markers CD4⫹, CD8⫹, TCR ␣␤⫹, TCR ␥␦⫹, CD8␣␤⫹, CD8␥␦⫹ was carried out by FACS after incubation with labeled mAbs specific to the correspondent cell surface marker. As shown in Table 1, inoculated SCID mice transferred with primed IEL exhibited significantly higher proportions of TCR ␣␤⫹, CD8⫹, and CD8␣␤⫹ than inoculated SCID mice that received unprimed or no (control) IEL (P ⬍ 0.01, P ⬍ 0.05, P ⬍ 0.01, respectively). Inoculated SCID mice transferred with unprimed IEL had higher proportions of CD8␣␤⫹ IEL than inoculated SCID mice given no (control) IEL (P ⬍ 0.01). Figure 2 shows the distribution of TCR ␣␤⫹ and CD8⫹ IEL in typical cell suspensions from inoculated SCID mice given primed, unprimed, or no (control) IEL. Higher numbers of CD8⫹ and TCR ␣␤⫹ were found in inoculated SCID mice transferred with primed (Figure 2C) IEL than in inoculated SCID mice that received unprimed (Figure 2B) or no (control, Figure 2A) IEL, P ⬍ 0.01. Discussion The results presented herein suggest to us that immunity against C parvum could be adoptively transferred with IEL and that IEL may have an important regulatory and/or effector function in mucosal responses to intestinal C parvum infection in adoptively transferred SCID mice. The significant increase of IEL numbers, including TCR ␣␤⫹, CD8⫹, and CD8␣␤⫹ T cells in recipient SCID mice, may indicate that transferred IEL populations functionally homed to intestinal sites and may have influenced responses to C parvum infection. Primed and unprimed IEL transfers were each associated with reduced fecal oocyst shedding in SCID mice compared with SCID mice that received no IEL. However, stronger resistance was demonstrated with primed IEL than unprimed IEL. Similar results were reported by McDonald et al13 involving primed THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

and unprimed mesenteric lymph node cells in SCID mice during infection with C muris, a closely related parasite.13 The reason(s) why unprimed IEL conferred protection comparable to that of primed IEL cannot be discerned from our study. A possible explanation for this phenomenon may be that the unprimed IEL became activated after initial antigen exposure, because IELs are predominantly resting memory cells.28 It is also likely that unprimed IEL transferred to SCID mice became responsive and/or functional and influenced existing IEL subsets to up-regulate other host mucosal responses leading to reduced oocyst shedding. Further studies are needed to define and elucidate the relative contributions of primed and unprimed IEL in protective immune response to C parvum infection in SCID mice. Although it has been suggested that TCR ␥␦⫹ IEL may be important in the recovery from infections caused by obligate intracellular parasite infections,17,28 during chronic cryptosporidiosis we did not observe expansion of TCR ␥␦⫹ IEL in SCID mice transferred with either primed or unprimed IEL. It seems, however, that TCR ␥␦⫹ IEL may be stimulated early in response to C parvum infection in mice (Adjei & Enriquez, unpublished data). Another contributing factor may be that IEL are unable to differentiate into TCR ␥␦⫹. Because the expression of TCR ␥␦⫹ on IEL depends on a unique lineage of T cells,29 –31 including early fetal thymocytes and peripheral T cells, it is tempting to speculate that the absence of or decreased TCR ␥␦⫹ T cells may be caused by lack of peripheral T cell and thymocytes in SCID mice. Alternatively, it may be that TCR ␥␦⫹ T cells exhibit impaired homing capacities and that IEL may acquire TCR ␥␦⫹ expression after they have localized in the intestinal epithelium. Whether TCR ␥␦⫹ are of importance in the development of mucosal immunity to C parvum remains to be investigated. Within the IEL populations, TCR ␣␤⫹, CD4⫹, CD8⫹, CD4␣␤⫹, and CD8␣␤⫹ T cells have been shown to have roles in host immunity against enteric invasion by pathogens.32–37 Transfer of IEL from immune BALB/cJ mice to recipient SCID mice resulted in increased proportions of TCR ␣␤⫹, CD4⫹, and CD8⫹ IEL and reduction of C muris infection.32 Buzoni-Gatel et 307

Adoptive Transfer of Immunity

ferred with primed IEL compared with inoculated SCID mice that were given unprimed or no (control) IEL. The increased expansion of TCR ␣␤⫹, CD8⫹, and CD8␣␤⫹ IEL could be explained by the occurrence of local T-cell-mediated responses during this enteric disease. Previous reports have shown that CD8␣␤⫹, TCR ␣␤⫹, CD4⫹, CD8⫹, CD4␣␤⫹ IEL isolated from mice with T gondii or C muris infections were activated in response to T gondii- and C muris-infected enterocytes, leading to a reduced number of cysts in the brain and oocysts in feces, respectively.14,32–34,36,37,39 Although in our studies we did not attempt selective depletion of IEL subsets or address whether enriched CD8␣␤⫹, TCR ␣␤⫹, CD4⫹, CD8⫹, or CD4␣␤⫹ IEL in SCID mice are responsible for the lower oocyst shedding, it is possible that in the absence of systemic T and B cells, TCR ␣␤⫹, CD8⫹, and CD8␣␤⫹ IEL may play a role in protective immune response against C parvum when transferred to SCID mice. Whether the IEL populations studied represented cells that underwent intra- or extrathymic T cell development is still unclear.38,40 Nevertheless, the data herein suggest to us that regardless of the origin, intestinally derived IEL may be functional, may have triggered functional effector cells, and/or were preferentially stimulated after C parvum infection. In summary, we conclude that primed IEL from immunocompetent mice may influence protective mucosal response against cryptosporidiosis when transferred into SCID mice. In addition, the increased percentage of TCR ␣␤⫹, and CD8⫹, CD8␣␤⫹ IEL in recipient SCID mice may reflect mucosal cell populations involved in these responses during chronic C parvum infection. Studies are now in progress to assess the potential function of these IEL as well as their cytokine secretion pattern in SCID mice during cryptosporidiosis. Acknowledgments

Figure 2. FACS distribution of CD8⫹ and T-cell receptor ␣␤⫹ IEL from SCID mice adoptively transferred with sterile PBS (control), unprimed or primed IEL 72 days after oral inoculation with 1 ⫻ 106 C parvum oocysts. Each contour line represent 10% of cell populations. (A) Inoculated SCID mice transferred with PBS (control). (B) Inoculated SCID mice transferred with unprimed IEL. (C) Inoculated SCID mice transferred with primed IEL.

al36 and Chardes et al33 noted an increase of TCR ␣␤⫹, CD8␣␤⫹, and CD8⫹ IEL and decreased parasite burden during infection with Toxoplasma gondii. A similar role of CD8⫹, CD8␣␤⫹ IEL was described during intestinal Listeria monocytogenes infection.38 In the present study, increased expansion of TCR ␣␤⫹, CD8⫹, and CD8␣␤⫹ IEL T cells and a lower oocyst shedding was observed in inoculated SCID mice trans308

The excellent technical assistance of Lisa Delsid, Brian Curran, and Barbara Carolus is greatly appreciated. References 1. Fayer R, Speer CA, Dubey JP. The general biology of Cryptosporidium. In: Fayer R, editor. Cryptosporidium and cryptosporidiosis. 1st ed. New York: CRC Press; 1997. p. 1– 42. 2. Blagburn BL, Soave R. Prophylaxis and chemotherapy: human and animal. In: Fayer R, editor. Cryptosporidium and cryptosporidiosis. 1st ed. New York: CRC Press; 1997. p. 111–28. 3. Riggs MW. Immunology: host response and development of passive immunotherapy and vaccines. In: Fayer R, editor. Cryptosporidium and cryptosporidiosis. 1st ed. New York: CRC Press; 1997. p. 129 – 62. 4. Aguirre SA, Mason PH, Perryman LE. Susceptibility of major histocompability complex (MHC) Class I-and MHC

November 2000 Volume 320 Number 5

Adjei et al

5. 6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

class II-deficient mice to Cryptosporidium parvum infection. Infect Immun 1994;62:697–9. Current WL, Bick PH. Immunobiology of Cryptosporidium spp. Pathol Immunopathol Res 1989;8:141– 60. Arrowood MJ, Mead JR, Mahrt JL, et al. Effects of immune colostrum and orally administered anti-sporozoite monoclonal antibodies on the outcome of Cryptosporidium parvum infections in neonatal mice. Infect Immun 1989;57: 2283– 8. Bjorneby JM, Hunsaker BD, Riggs MW, et al. Monoclonal antibody immunotherapy in nude mice persistently infected with Cryptosporidium parvum. Infect Immun 1991;59:1172– 6. Darban H, Enriquez FJ, Sterling CR, et al. Cryptosporidiosis facilitated by murine retroviral infection with LP-BM5. J Infect Dis 1991;164:741–5. McDonald V, Deer R, Uni S, et al. Immune responses to Cryptosporidium muris and Cryptosporidium parvum in adult immunocompetent and immunocompromised (nude and SCID) mice. Infect Immun 1992;60:3325–31. Rasmussen KR, Healey MC. Experimental Cryptosporidium parvum infection in immunosuppressed adult mice. Infect Immun 1992;60:1648 –52. Rohlman VC, Kuhls TL, Mosier DA, et al. Cryptosporidium parvum infection after abrogation of natural killer cell activity in normal and severe combined immunodeficiency mice J Parasitol 1993;79:295–7. Gardner AL, Roche JK, Weikel CS, et al. Intestinal cryptosporidiosis: pathophysiologic alterations and specific cellular and humoral immune responses in RNU/⫹ and RNU/ RNU (athymic) rats. Am J Trop Med Hyg 1991;44:49 – 62. McDonald V, Robinson HA, Kelly JP, et al. Cryptosporidium muris in adult mice: adoptive transfer of immunity and protective roles of CD4⫹ versus CD8⫹ cells. Infect Immun 1994;62:2289 –94. Culshaw RJ, Bancroft GJ, McDonald V. Gut intraepithelial lymphocytes induce immunity against Cryptosporidium infection through a mechanism involving gamma interferon production. Infect Immun 1997;65:3074 –9. Pasquali P, Fayer R, Almeria S, et al. Lymphocyte dynamic patterns in cattle during a primary infection with Cryptosporidium parvum. J Parasitol 1997;83:247–50. Wyatt CR, Brackett EJ, Perryman LE, et al. Activation of intestinal intraepithelial T lymphocytes in calves infected with Cryptosporidium parvum. Infect Immun 1997;65:185– 90. Kaufmann SHE. Gamma/delta and other unconventional T lymphocytes: what do they see and what do they do? Proc Natl Acad Sci U S A 1996;93:2272–9. Barrett TA, Gajewski TF, Danielpour D, et al. Differential function of intestinal intraepithelial lymphocyte subset. J Immunol 1992;149:1124 –30. Guy-Grand D, Cerf-Bensussan N, Malissen B, et al. Two gut intraepithelial CD8⫹ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation. J Exp Med 1991;173:471– 81. Guy-Grand D, Cuenod-Jabri B, Malassis-Seris M, et al. Complexity of the mouse gut T cell immune system: identification of two distinct natural killer T cell intraepithelial lineages. Eur J Immunol 1996;26:2248 –56. Guy-Grand D, DiSanto JP, Henchoz P, et al. Small bowel enteropathy: role of intraepithelial lymphocytes and cytokines (IL-12, IFN-gamma, TNF) in the induction of epithelial cell death and renewal. Eur J Immunol 1998;28:730 – 44. Lefrancois L, Fuller B, Huleatt JW, et al. On the front lines: intraepithelial lymphocytes as primary effectors of in-

THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

23.

24.

25.

26.

27.

28.

29.

30.

31. 32.

33.

34.

35.

36.

37.

38.

39. 40.

testinal immunity. Springer Semin Immunopathol 1997;18: 463–75. Adjei AA, Jones JT, Riggs MW, et al. Evidence of thymusindependent local and systemic antibody responses to Cryptosporidium parvum infection in nude mice. Infect Immun 1999;67:3947–51. Huang DS, Lopez MC, Wang JY, et al. Alterations of the immune system due to Cryptosporidium parvum infection in normal mice. Cell Immunol 1996;173:176 – 82. Ernst PB, Clark DA, Rosenthal KL. Detection and characterization of cytotoxic T lymphocyte precursors in the murine intestinal intraepithelial leukocyte population. J Immunol 1986;136:2121– 6. Ernst PB, Befus AD, Dyck N, et al. Cell separation: methods and selected applications. Pretlow TG, Pretlow TP, editors. 2nd ed. New York: Academic Press; 1987. p. 141–50. Adjei AA, Jones JT, Enriquez FJ. Differential intraepithelial lymphocyte phenotypes following Cryptosporidium parvum challenge in susceptible and resistant athymic strains of mice. Parasitol Int; in press. McGhee JR, Hiroshi K. The mucosal immune system. In: Paul E, editor. Fundamental immunology. 4th ed. Philadelphia: Lippincott-Raven; 1999. p. 909 – 45. Bluestone JA, Matis LA. TCR gamma delta cells minor redundant T cell subset or specialized immune system component? J Immunol 1989;142:1785– 8. Janeway CA, Jones B, Hayday A. Specificity and function of T cells bearing gamma delta receptors. Immunol Today 1988;9:73– 6. Janeway CA. Frontiers of the immune system. Nature 1988; 333:804 – 6. McDonald V, Robinson HA, Kelly JP, et al. Immunity to Cryptosporidium muris infection in mice is expressed through CD4⫹ intraepithelial lymphocytes. Infect Immun 1996;64:2556 – 62. Chardès T, Buzoni-Gatel D, Lepage A, et al. Toxoplasma gondii oral infection induces specific cytotoxic CD8␣/␤⫹ Thy-1⫹ gut intraepithelial lymphocytes, lytic for parasiteinfected enterocytes. J Immunol 1994;153:4596 – 603. Lepage AC, Buzoni-Gatel D, Bout DT, et al. Gut-derived intraepithelial lymphocytes induce long term immunity against Toxoplasma gondii. J Immunol 1998;161:4902– 8. Emoto M, Emoto Y, Kaufmann SH. Development of CD8 alpha/beta⫹ TCR intestinal intraepithelial lymphocytes in athymic nu/nu mice and participation of regional immune responses. Immunol 1996;88:531– 6. Buzoni-Gatel D, Lepage AC, Dimier-Poisson IH, et al. Adoptive transfer of gut intraepithelial lymphocytes protects against murine infection with Toxoplasma gondii. J Immunol 1997;158:5883–9. Buzoni-Gatel D, Debbabi H, Moretto M, et al. Intraepithelial lymphocytes traffic to the intestine and enhance resistance to Toxoplasma gondii oral infection. J Immunol 1999;162:5846 –52. Emoto M, Neuhaus O, Emoto Y, et al. Influence of ␤2microglobulin expression on gamma interferon secretion and target cell lysis by intraepithelial lymphocytes during intestinal Listeria monocytogenes infection. Infect Immun 1996; 64:569 –75. Poussier P, Julius M. Intestinal intraepithelial lymphocytes: the plot thickens. J Exp Med 1994;180:1185–9. Rocha B, Vassalli P, Guy-Grand D. The extrathymic T-cell development pathway. Immunol Today 1992;13:449 –54.

309