Immunology 1992 77 38-42

In vivo inhibition by a monoclonal antibody to CD4+ T cells of humoral and cellular immunity in sheep H. S. GILL, D. L. WATSON* & M. R. BRANDONt Department of Animal Science, University of New England and *CSIRO Division of Animal Health, Pastoral Research Laboratory, Armidale, New South Wales, and tCentre for Animal Biotechnology, University of Melbourne, Parkville, Victoria, Australia Acceptedfor publication 20 April 1992

SUMMARY The ability of intravenously injected anti-CD4 and anti-CD8 monoclonal antibody (mAb) to deplete specific lymphocyte subsets in vivo and their effects on antibody responses to ovalbumin (OVA) and Brucella abortus, and skin reactivity to T-cell mitogens was examined in merino lambs. Repeated administration of anti-CD4 or anti-CD8 mAb caused a specific and sustained depletion of target cells from peripheral blood. Anti-CD4 mAb significantly inhibited the in vivo antibody response to OVA but had no effect on the antibody response to LPS of B. abortus. In contrast, antibody responses to both OVA and B. abortus lipopolysaccharides (LPS) remained unaffected in lambs depleted of their CD8+ T lymphocytes. These results confirm the T-cell dependence and independence of antibody responses to OVA and LPS, respectively. Skin reactions elicited by intradermal injections of phytohaemagglutinin (PHA) and concanavalin A (Con A) were also significantly suppressed in lambs depleted of their CD4+ T cells, but treatment with 'anti-CD8 mAb had no effect on skin responsiveness. Together, these results suggest that mAb can be extremely effective at selectively depleting lymphocyte subsets in vivo and can be used for studying various aspects of immunoregulation and immunity in sheep.

populations in pathogenesis, and in the development of immu-

INTRODUCTION Over the last decade, athymic mice and inbred rodents have been extensively used in cell-transfer studies to define lymphocyte populations involved in the regulation of protective immune responses and their possible mode of action. However, in domestic animals similar studies have not been possible due to unavailability of inbred lines or athymic strains. The development of monoclonal antibody (mAb) to lymphocyte surface markers in domestic animals'-5 has provided new opportunities to study these problems in vivo and in vitro. Selective depletion of lymphocyte subsets in vivo using mAb has been used to examine the contribution of various lymphocyte subsets to allograft rejection,6-7 auto-immune reactions8-'0 and infectious diseases in laboratory rodents."-"4 These studies highlight the possibility of modulating immune responsiveness by the selective manipulation of T-cell subpopulations. This method also has considerable potential for investigating the role of different lymphocyte

nity to infectious diseases in domestic livestock. Two major subpopulations of T lymphocytes identified in sheep are SBU T4+ and SBU T8+ cells, the homologue of the human CD4+ and CD8+ cells, respectively.2 CD4+ T cells comprise approximately 20% of peripheral blood mononuclear cells (PBMC), 80% of thymocytes and 50% of lymphocytes in efferent lymph. CD4+ thymocytes co-express CD8 antigen, whereas CD4+ PBMC do not express CD8 antigen. CD8+ T cells comprise 12% of PBMC, 80% of thymocytes and 15% of lymphocytes in efferent lymph.'5 Cells expressing CD4 antigen recognize specific antigen in association with major histocompatibility complex (MHC) class II molecules and function as helper/inducer cells, whereas cells expressing CD8 antigen recognize antigen in the context of MHC class I molecules and function as suppressor/cytotoxic cells.'5"16 The major objectives of the present study were to determine whether depletion of CD4+ and CD8+ T lymphocytes in vivo was practicable in sheep and, if so, to examine the effect of depleting CD4+ or CD8 + cells on antibody responses to ovalbumin (OVA) and Brucella abortus and skin reactivity to T-cell mitogens.

Abbreviations: APC, antigen-presenting cells; Con A, concanavalin A; ELISA, enzyme-linked immunosorbent assay; 1g, immunoglobulin; i.v., intravenous; LPS, lipopolysaccharide; mAb, monoclonal antibody; MHC, major histocompatibility complex; NMIg, normal mouse immunoglobulin; OVA, ovalbumin; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PBST, PBS-Tween 20 (0 05%); PHA, phytohaemagglutinin. Correspondence: Dr H. S. Gill, Dept. of Animal Science, University of New England, Armidale, NSW 2351, Australia.

MATERIALS AND METHODS Animals Fine-wool merino lambs, genetically resistant to Haemonchus contortus,'7 born and raised on pasture were used. Lambs were

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In vivo inhibition of immunity by anti-CD4 mAb in sheep Table 1. Monoclonal antibodies used in treatment and for analysis of lymphocyte phenotypes

Antigen specificity SBU-T4 (CD4)

Hybridoma (mAb clone) Isotype Antigen distribution 44-38

IgG2a Lymphocytes that are CD8-, SBU-T19-; absent from B

SBU-T8 (CD8)

38 65

lymphocytes IgG2a Lymphocytes that are CD4-, SBU-TI9-; absent from B lymphocytes

housed indoors on slatted floors and were fed on lucerne chaff and commercial pellets. All animals had experienced two artificial infections with H. contortus and were infected with H. contortus at the time of experiment. Lambs weighed 18-21 kg and were 5-6 months old at the start of the experiment.

T-cell-specific mAb The mAb used in this study (Table 1) were produced and characterized at the Centre for Animal Biotechnology (University of Melbourne, Parkville, Australia) as previously described.2

Preparation of PBMC Blood was collected by jugular venipuncture into 10-ml evacuated tubes containing preservative-free heparin (Becton Dickinson, Rutherford, NJ) and centrifuged at 1800g rpm for 20 min at room temperature. The buffy-coat was harvested, diluted 1 :2 in phosphate-buffered saline (PBS), layered onto Ficoll-Paque (Pharmacia, Uppsala, Sweden) and centrifuged at 800 g for 40 min. The mononuclear cells at the interface were collected, washed three times in PBS and then resuspended in HBSS containing 1% bovine serum albumin (Sigma) at a concentration of 2 x 106 cells/mi. Cell viability was determined using trypan blue exclusion and was over 95% in all cases. One hundred microlitre samples of cell suspensions were then used to prepare cytospots with a cytocentrifuge. Cytospots were air dried for 2 hr at room temperature, then fixed in acetone/chloroform (1: 1) for 10 min. After drying at room temperature, slides were wrapped in plastic bags to exclude air and stored at -20° until used.

Characterization of lymphocyte subsets in peripheral blood The immunoperoxidase technique was used to enumerate lymphocyte subsets. All incubations were done at room temperature. Cytospots were brought to room temperature, rehydrated in PBS for 10 min and incubated with 0-5% hydrogen peroxide in 20% methanol for 15 min to block endogenous peroxidase activity. Cytospots were then reacted with the optimal dilution of appropriate mAb for 45 min, washed in PBS ( x 3) and incubated with peroxidase-conjugated rabbit antimouse immunoglobulin (Dakopatts, Glostrup, Denmark). After incubation for 45 min, slides were incubated with 0-06% diaminobenzidine hydrochloride (Sigma), 0-1% H202 (30% w/ v) in citrate buffer, pH 5 0 for 10 min. The reaction was stopped by immersing slides in distilled water for 5 seconds. Slides were

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subsequently washed in PBS, counter stained with haematoxylin, rinsed in tap water, dehydrated, cleared in xylene and mounted in DPX. Specificity for immunostaining was determined by omitting the primary mAb. The percentage of immunoperoxidase positive cells was determined by counting a total of 300 cells/sample at x 400 magnification. Cells were also stained with peroxidase-conjugated rabbit anti-mouse immunoglobulin to detect administered mAb on target cells. This procedure showed no detectable mouse immunoglobulin on target cell surfaces, and indicated that there was no masking of surface antigens on these cells. Immunization Lambs were immunized by intramuscular injection (left medial thigh) with a mixture of 20 mg OVA and 1010 killed B. abortus cells (washed agglutinating suspension, CSL) in 1 ml sterile PBS on Day 0. Treatment with mAb Lambs were injected i.v. with 2 mg of anti-CD4+ or anti-CD8+ T-cell mAb on Days -3, -1, 3, 7 and 10, and I mg mAb on Days 14, 18 and 22 after immunization with OVA and B. abortus (Day 0). Control lambs received normal mouse immunoglobulin (NMIg) according to the schedule used for mAb treatment. Assay for antibodies to OVA and B. abortus Anti-OVA antibody levels in sera were assessed by an ELISA as previously described.'8 Anti-Brucella lipopolysaccharide (LPS) antibodies were measured using an ELISA which was adapted from bovine anti-Brucella ELISA19 for which LPS extracted from the cell wall of B. abortus was the coating antigen. Additionally, some sera were treated with 2-mercaptoethanol prior to assay for assessment of IgM antibody; monospecific anti-IgM and anti-IgG were used for some assays.

Skin reactivity to mitogens Skin test sites were prepared by closely clipping the sides of lambs with electric small animal clippers and surface sterilizing the skin with 70% alcohol. A test pattern of three rows and as many columns (7 cm apart) was then constructed using a felt pen. The skin test sites in the first row were intradermally injected with 10 ,ul phytohaemagglutinin (PHA) (Difco, Detroit, MI) using a 27 gauge needle. Rows 2 and 3 received PBS (control) and 10 ,ug concanavalin A (Con A) (Calbiochem, La Jolla, CA), respectively. All agents were injected in 100 p1 volumes and PBS was used as the diluent. Skin thickness was measured at 0 and 24 hr after inoculation with a micrometer gauge, calibrated to 0-01 mm. Skin reactivity was expressed as the difference between pre- and post-inoculation skin thickness. Experimental design Twelve lambs were randomly divided into three groups. Lambs in Group 1, (n = 3) were treated with anti-CD4 mAb and lambs in Group 2 (n = 3) were treated with anti-CD8 mAb. Control lambs (group 3, n = 6) received NMIg. All the lambs were immunized with OVA and Brucella cells on Day 0. The skin reactivity of all lambs to T-cell mitogens was determined on Day 23. Blood samples for isolation of PBMC and for sera were collected at regular intervals throughout the experiment. Sera were stored at - 20° until used.

H. S. Gill, D. L. Watson & M. R. Brandon

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Time (days) Figure 1. CD4+ T lymphocytes (%) in the peripheral blood of control lambs (0) and lambs treated with anti-CD4 (U) or anti-CD8 (0) mAb. Lambs were treated (i.v.) with 2 mg of mAb on Days -3, -1, 3, 7 and 10, and 1 mg mAb on Days 14, 18 and 22. Control lambs received NMIg.

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Days after immunization Figure 3. Antibody response (mean + SEM) to OVA in control lambs (0) and lambs depleted of their CD4+ (U) or CD8+ (U) T cells. Cell depletion was accomplished by treating lambs with mAb to the appropriate determinant on Days -3, 1, 3, 7, 10, 14, 18 and 22. Lambs were immunized with a mixture of 20 mg OVA and 1010 killed B. abortus cells in 1 ml PBS on Day 0, and the antibody levels were determined using an ELISA. Probability versus control lambs treated with NMIg; (a) P