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Non-immune erythrocyte rosette formation of bovine peripheral blood and thymus lymphocytes
Comp. lmmun. Microbiol. infect. Dis., Vol.4, No. 1, pp. 75 86, 1981
0147 9571/81/010075-12 $02.00/0 ~ 1981 PergamonPressLtd.
Printed in Great Britain.
N O N - I M M U N E E R Y T H R O C Y T E ROSETTE F O R M A T I O N OF BOVINE P E R I P H E R A L BLOOD A N D THYMUS LYMPHOCYTES HARLAND W . RENSHAW Department of Veterinary Microbiology and Parasitology, Texas Agricultural Experiment Station, College of Veterinary Medicine, Texas A&M Universit L College Station, TX 7784K U.S.A. Abstract--An E-rosetting reaction is described which gave 92.1°~_+ 2.4 (mean_+ S.D.) E-rosettes with bovine fetal thymocytes and 48.2~o + 8.4 with with bovine peripheral blood leukocyte (PBL) preparations. Both culture conditions and culture medium were critical factors in obtaining maximal and reproducible E-rosette numbers. Optimum rosette formation occurred when bovine PBL and neuraminidase treated sheep erythrocytes (nSRBC) were reacted in L-15 culture medium supplemented with 10°~, fetal calf serum (FCS). Other media including 100~o FCS, M E M with 10~o FCS, and RPMI-1640 with 100/oFCS were less satisfactory. Cultural conditions found to be optimal for enumeration of bovine E-rosettes are similar to those reported as optimal for detection of human T cells. The specificity of rosette formation by bovine thymus derived (T) lymphocytes was shown by demonstration of (1) rosettes and surface membrane immunoglobulins (mIg) on different cells in PBL, (2) rosette formation by the majority of fetal thymocytes, and (3) no inhibition of rosette formation by anti-immunoglobulin serum. Using the E-rosette and mIg assays for presumptive bovine T and B lymphocytes, respectively, it was possible to differentiate from 57.5 to 900 (75.2°0 _+9.3) of cells in bovine PBL preparations, and from 90.2 to 97.50/o (94.2°% _+2.1) of cells in bovine fetal thymocyte preparations into T and B cells. Key words: Non-immune erythrocyte rosettes, E-rosettes, T cells, T lymphocytes, bovine T and B
lymphocytes LA FORMATION LYMPHOCYTES
DE ROSETTES DE BOVIN
NON
IMMUNES
ISSUS DU
DE
SANG
PERIPHERIQUES OU DU THYMUS R o s u m ~ - La reaction de rosettes E decrite donne 92,1"~_+ 2,4 de rosettes avec les lymphocytes du thymus et 48,2%o_+8,4 avec les preparations leucocytaires issues du sang peripherique (PBL). Les conditions et le milieu de culture sont des facteurs importants pour l'obtention d'un hombre maximum de rosettes E et la reproductibilite de la reaction. La quantit6 optimale de rosettes a 6re obtenue quand la PBL bovine et des ~rythrocytes de mouton impregnes de neuraminidase (nSRBC) ont 6te places dans un milieu de culture L-15 additionne de 10'~,ide serum foetal de veau (FSC). D'autres milieux, dont le FSC seul, le MEM avec 10'~,~FCS, et le RPMI-1640 avec 10!i~ FSC laissaient ~i d6sirer. Les conditions de culture qui ont ete decrites comme optimales pour l'obtention des rosettes E bovines, sont semblables fi celles rapportees comme optimales pour les cellules T humaines. Trois points ont permis de d6montrer la spbcificit6 des cellules du thymus formant des rosettes: II existe des immunoglobulines M sur la membrane de quelques cellules issues du sang peripherique. (2) La majorite des thymocytes foetaux forment des rosettes. (3) la formation des rosettes n'est pas inhibee par un s~rum antiimmunoglobuline. En utilisant les rosettes E el les dosages des immunoglobulines M pour identifier, respectivement, les lymphocytes T et les lymphocytes B, nous avons pu caracteriser 57,5 fi 90')o (75,27 _+9,3) des cellules du sang p6ripherique et 90,2 fi 97, 5"% (94,2~o _+2,1) des cellules thymiques en lymphocytes T ou B. Mots-clef~: Rosettes erythrocytes non-immunes, rosettes E, cellules T~ lymphocytes T, lymphocytes bovins T et B INTRODUCTION
It is now generally accepted that in all mammalian species there are two distinct classes of lymphocytes, each with different functional activities and cell surface membrane 75
76
HARLAND W. RENSHAW
receptors. The T lymphocytes of many species have been shown to form non-immune erythrocyte (E) rosettes with heterologous erythrocytes, and the B lymphocytes to bind fluorescein-labeled antiserum to immunoglobulins of the species involved. Several groups have recognized E-rosette formation with bovine lymphocytes incubated under appropriate conditions with suitably prepared sheep red blood cells (SRBC). Although each group concluded that E-rosettes could be used as a marker of bovine T cells, there were significant differences in the percentage of E-rosette forming lymphocytes reportedly found in peripheral blood and thymus. Higgins and Stack suggested that only a limited population of bovine T cells, possibly immature bovine T cells, form E rosettes and that this sub-population is either absent or present in low percentages in peripheral blood of calves and adults [1]. In several studies it has been speculated that even under the experimental conditions found to be optimum that not all bovine T cells formed E rosettes [1 3]. Studies of E-rosette forming cells in other species suggest that slight variations in procedural techniques can account for dramatic differences in percentages of E-rosette forming lymphocytes found in the peripheral blood or organs of normal subjects. Different treatments of SRBC or additives to the rosetting medium have been used to enhance rosette formation by presumptive bovine T lymphocytes [3 8]. Considerable confusion exists about the reliability of the various different methods described for demonstrating presumptive bovine T lymphocytes because investigators have not always adequately demonstrated the specificity of their E-rosetting reaction. In some cases, the specificity of the E-rosetting reaction has been proposed as adequate if E-rosette forming cells were found in percentages that closely approximated what was thought to be the expected distribution of T cells in bovine peripheral blood and thymus lymphocyte preparations. The expected distribution being extrapolated from previously published data about T-cell distribution in humans and laboratory animals. If an E-rosette forming reaction is to be considered as a T-cell marker for cattle the rosette-forming reaction should: (1) occur with the majority of thymus cells and with a large enough percentage of peripheral blood lymphocytes as to exclude reaction by specific immunoglobulin receptors, (2) occur with a cell population separate from that bearing detectable immunoglobulin determinants, and (3) not be inhibited by anti-immunoglobulin serum [9]. To date none of the described methods for detecting presumptive bovine T lymphocytes has been shown to completely fulfill each of these requirements. In a previous study, techniques were evaluated for identifying sub-populations of bovine peripheral blood lymphocytes [7]. Presumptive bovine T cells were identified in peripheral blood lymphocyte preparations using conditions similar to those reported as optimal for human cells. Evidence will be presented in this report that (1) this E-rosette forming reaction fulfills each of the above indicated requirements and is therefore a reliable marker for bovine T lymphocytes, and (2) both culture conditions and media are critical factors in the non-immune E-rosette technique with bovine lymphocytes.
MATERIALS AND METHODS Lymphocyte sources
Heparinized blood samples (15 U/ml) were collected by jugular venipuncture from Holstein Friesian or cross-bred beef cattle of different ages. All animals were fed a
Non-immunerosetteformationof bovine lymphocytes
77
standard ration and were clinically normal. Bovine fetal thymus tissue was obtained at a local abattoir. Normal appearing fetuses, 7-9 months of estimated gestational age, were obtained from beef type cattle passed for human consumption by government inspection.
Sheep erythrocytes (SRBC) Venous blood from clinically healthy sheep was collected into sterile Alsever's solution pH 6.1 [10]. Collected sheep blood was stored as a 50~ suspension in Alsever's solution at 4'C, and used in the E-rosette assay between 1 and 3 weeks afer collection. Before use, SRBC were washed twice in sterile physiologic saline (PS), recovered between washings by centrifugation at 400 x g for 10 min at 22'~C, and resuspended to 0.5% in the appropriate medium.
Neuraminidase treatment of SRBC The SRBC used in the E-rosette assay were prepared for use as described above or after neuraminidase treatment with Vibrio cholerae filtrate (Sigma Chemical Co., St Louis, MO) that was reconstituted (0.05 U/ml) with PS. A mixture of 1 vol of packed washed SRBC and 5 vol of reconstituted filtrate were incubated at 37~C for 60 min. The treated cells were washed twice in the appropriate medium.
Media and sera The same lot of fetal calf serum (FCS) was used throughout this study (lot 086312, Grand Island Biological Co., Grand Island, NY). The FCS was inactivated at 56°C for 30min and absorbed at 37~:C for l hr against an equal volume of packed SRBC. Fluorescein-conjugated (FITC) rabbit antiserum prepared against the 7S globulin fraction of bovine serum (RaBIg) was purchased (Miles Laboratories, Inc., Elkhart, IN). Several commercially available media (Grand Island Biological Co., Grand Island, NY), including Leibovitz medium (L-15), RPMI 1640, and Eagle's minimal essential medium (MEM), all at pH 7.4, were compared for their capacity to support E-rosette formation.
Peripheral blood lymphocyte isolation Bovine peripheral blood lymphocytes (PBL were isolated using a modified version of the method of B6yum [11]. Heparinized blood (15 ml) was mixed with an equal volume of L-15, or another appropriate medium containing 15 U of heparin/ml in a 50-ml conicalbottom plastic screw-top centrifuge tube. Sucrose copolymer/sodium diatrizoate (24 parts of 9~o w/v aqueous solution of Ficoll 400, Pharmacia Fine Chemicals, Uppsala, Sweden, 10 parts of 33.9% w/v aqueous solution of Hypaque, Winthrop Laboratories, NY, with specific gravity adjusted to 1.077) solution (20 ml) was layered beneath the blood-media mixture, and tubes were centrifuged at 400 x g for 40 min at 22°C. The mononuclear cell layer at the gradient interface was collected, contaminating erythrocytes were removed by lysis with 3 vol of 0.87~o NH4C1 [2], and the leukocytes recovered by centrifugation at 400 x g for 10 min at 22"C. The cells were washed twice in PS with heparin (15 U/ml), and adjusted to 15 x 106 lymphocytes/ml. Differential leukocyte counts were performed on Wright's stained smears. The cells were then washed two more times in the appropriate test medium.
78
HARLAND W. RENSHAW
Cell suspensions free of erythrocytes contained approximately 91~o lymphocytes O . .l . .9. °Jo/ -+ 5.8" range 82-99%) and 5 ~ monocytes (5.1 o~o -+ 4.4; range 1-15%). Greater than 95",~i of the cell population was capable of excluding 0.1% trypan blue in all experiments.
Thymocyte isolation Bovine fetal thymus tissue was transported on ice in phosphate-buffered saline (pH 7.4). The thymic tissue was dissected free of connective tissue and blood vessels, minced, and gently squeezed with a rubber policeman through a 120 mesh stainless sieve into PS with heparin (15 U/ml). The suspension was centrifuged at 4 0 0 x g for 10 min at 2 2 C , then resuspended in 3 vol of 0,87°,o NHaC1 to remove contaminating erythrocytes. The thymocytes were collected by centrifugation, washed twice in L- 15 and resuspended in L- 15 with 100~i FCS at 1 5 x 1 0 ~' thymocytes/ml. The final cell suspension contained approximately 97°;; lymphocytes (97.4°o_+2.4; range 93 100'~'o) and greater than 96°~i of the cell population was capable of excluding trypan blue.
Bovine E-rosette test A suspension of 2.25 x 106 PBL or thymocytes in 0.15 ml of L- 15 or another appropriate medium with lO',!Jo FCS was combined with 0.3 ml of a 0.5°J~i suspension of either neuraminidase treated (nSRBC) or untreated SRBC in L-15 or another appropriate medium with 10~o FCS. The cell mixture was incubated for 5 min in a 37~'C water bath, centrifuged at 200 x g for 5 rain at 2 2 C , and then incubated in an ice water bath (OC) for 24 hr. The cell pellet was resuspended and a drop of 0.21!,~itrypan blue added. An aliquot of the cell suspension was then added to a pre-chilled (4:'C) Neubauer counting chamber and t h e percentage of viable leukocytes binding two or more SRBC determined microscopically at 440 x . At least 200 mononuclear cells were examined for E-rosette formation for each sample. Cell counts were made between 1 and 10 min after placing the cell suspension in the counting chamber. The percentage of E-rosette forming cells with two or more SRBC attached was determined.
lmmunofluorescence test (IFT)./'or immunoglobulin-bearing l)'mpho~3,tes A suspension of 3 x 106 PBL or thymocytes in 0.2 ml L-15 was mixed with 0.2 ml o f a 1 : 10 dilution of FITC-labelled RaBIg, incubated at 4°C for 30 min with occasional gentle shaking, washed three times with L-15 at 4°C, and recovered between each wash by centrifugation at 400 x g for 5 min. The cell pellet was resuspended in several drops of glycerol: PBS (1 : 1) mounting medium and examined by fluorescence microscopy at 440 x using a fluorescence microscope equipped with mercury vapor lamp illumination (Carl Zeiss, NY). Four hundred cells were examined under both incandescent light and ultraviolet (u.v.). Cell membrane bound immunoglobulin (mIg) usually appeared as an homogeneous speckled distribution over the entire cell or as 'caps'. For simultaneous detection of E-rosettes and mIg-bearing lymphocytes the bovine cell preparations were reacted with FITC-labelled RaBIg as described above. Subsequently, to allow formation of E-rosettes, 0.1 ml of labelled leukocytes was combined with 0.2 ml of a 0.5% suspension of nSRBC in L-15 with 10% FCS. The labelled bovine leukocyte-nSRBC mixture was incubated for 5 rain at 37°C, pelleted by centrifugation at 200 x g at 22°C for 5 min, and
2.5
5.3
2.0 2.0 3.0 6.5 5.5 8.0 5.0 8.5 7.0
2-4
2.0-25.0
6.5
9.3
0.0 1.0 5.0 18.0 8.0 9.0 11.5 16.5 14.5
/> 5
8.7
14.6
2.0 3.0 8.0 24.5 13.5 17.0 16.5 25.0 21.5
total
L - 1 5 + 10% FCS with SRBC§
2.1
11.9
11.5 12.5 11.5 9.0 16.5 12.0 10.0 12.5 12.0
2 4
35.5 61.0
9.3
36.3
24.0 27.0 33.0 49.5 27.0 39.0 39.5 38.5 49.0
/> 5
8.4
48.2
35.5 39.5 44.5 58.5 43.5 51.0 49.5 51.0 61.0
total
L - 1 5 + 10% FCS with nSRBCII
1.7
5.0
5.0 3.0 4.0 6.5 7.5 4.5 0.5 2.5 5.5
2 4
20.0 55.0
11.1
33.2
21.0 17.0 28.0 48.5 34.0 40.5 30.0 30.5 49.0
~>5
12.0
38.2
26.0 20.0 32.0 55.0 41.5 45.0 36.0 32.5 54.5
total
100% FCS with nSRBC
2.8
7.4
6.0 9.0 4.0 9.0 7.5 11.0 7.0 3.0 10.5
2-4
7.5-43.5
11.0
14.0
3.0 2.0 3.5 27.0 8.5 20.0 15.5 13.5 33.0
/> 5
12.8
21.4
9.0 11.0 7.5 36.0 16.0 31.0 22.5 16.5 43.5
total
R P M I + 10% FCS with n S R B C
3.6
7.3
4.0 3.0 3.5 8.0 8.0 8.5 11.0 6.0 14.0
2 4
8.0-39.0
8.8
17.7
17.0 16.0 4.5 31.0 11.0 30.5 11.0 18.5 20.0
>/5
10.4
25.0
21.0 19.0 8.0 39.0 19.0 39.0 22.0 24.0 34.0
total
M E M + 10% FCS with n S R B C
* Standard rosette test performed as described in text, except with variation of media. L-15 = Leibovitz medium, FSC = fetal calf serum: RPMI = RPMI-1640; M E M = Eagle's minimal essential medium. Percentage of E-rosette forming cells expressed as cells with from 2 to 4, 5 or more, and total with 2 or more attached sheep erythrocytes. ++ mIg-Bearing cells are those cells showing m e m b r a n e fluorescence after treatment with fluorescein-conjugated rabbit anti-bovine immunoglobulin G; percentage of 400 cells. § SRBC = untreated sheep erythrocytes. II nSRBC = neuraminidase-treated sheep erythrocytes.
22.0-29.2
2.3
Range (mIg or total Erosettes)
27.1
1' 2 3 4 5 6 7 8 9
S.D.
22.0 28.5 27.5 25.0 29.2 27.0 28.5 26.8 29.0
Animal number
Mean
Lymphocytes with mlg~ (%)
PBL forming E-rosettes (%)*
Table 1. E-rosette formation by bovine PBL after incubating lymphocyte-SRBC pellets in different media
80
HARLAND W. RENSHAW
incubated for 24 hr at 0°C. The percentages of leukocytes with the different surface markers were determined for at least 200 mononuclear cells in a pre-chilled counting chamber under both incandescent light and u.v.
Influence of antiglobulin serum on E-rosette formation PBL were treated for 1 hr at 4°C with a 1/10 dilution of heat inactivated rabbit antibovine IgG containing 3.1 mg antibody protein per ml antiserum (Miles Laboratores Inc., Elkhart, IN). This concentration of antibody prevented immunofluorescent staining of immunoglobulin-bearing bovine lymphocytes. Control tubes were treated with heat inactivated normal rabbit serum at the same dilution. Cells were washed three times in L-15 with 10% FCS to remove excess serum before rosetting with nSRBC. RESULTS
Effect of incubating PBL-SRBC mixtures in different media Several different media were compared for their capacity to support formation of spontaneous, direct E-rosettes between bovine PBL and nSRBC. Bovine PBL and nSRBC were incubated for 5 min at 37°C, centrifuged at 200 × g for 5 min at 22°C, and incubated on ice (0c'C) for 2 4 h r with the following media: L - 1 5 + 1 0 ~ FCS, R P M I + I 0 ~ FCS, M E M + 10~ FCS, and 100~o FCS. Additionally, bovine PBL were mixed with untreated SRBC in L-15 + 10~ FCS. Parallel test results of E-rosette formation with bovine PBL in these various media are presented in Table 1. Optimum rosette formation occurred when neuraminidase treated SRBC were reacted with PBL in L-15 supplemented with 10~ FCS. Lymphocytes reacting in the medium gave the highest mean percentage of E-rosettes. For each animal tested the percentage of E-rosettes formed was highest with this medium. With the other media tested, the next highest percentage of rosettes formed when bovine PBL and nSRBC were incubated in 100% FCS. Clumping of lymphocytes occurred when some cell samples were incubated in 100~ FCS. This made interpretation of the E-rosette assay difficult when this medium was used. RPMI-1640+10~o FCS or M E M + 1 0 ~ FCS yielded the lowest number of rosette-forming cells. Treatment of SRBC with neuraminidase significantly enhanced rosette formation when bovine PBL-SRBC mixtures were incubated in L-15 + 10~ FCS (Table 1). In paralled with the rosette study, PBL were examined for mlg. Approximately 27~ of the PBL from the 9 clinically normal Holstein-Friesian cattle were positive for mlg (Table 1). Using the direct fluorescence test for detection of cells with mlg and the E-rosette test with L-15+ 10~ FCS and nSRBC it was possible to differentiate for 57.7 to 90~o (75.2~+9.3) of cells in bovine PBL preparations into presumptive T and B cells. Greater than 99~, of the bovine cells rosetting with nSRBC were identified as lymphocytes when Wright's stained smears were examined.
Effect of incubation temperature on E-rosette formation Bovine PBL and nSRBC mixtures in L - 1 5 + 10% FCS were incubated at either 37 or 2 2 C for 5 rain before centrifugation and incubation for 24 hr at WC. In all cases the percentages of E-rosette forming cells was higher at 37 than at 22°C (Table 2). If the PBLnSRBC pellets were held at either 22 or 37~C for 24 hr, rather than at 0"C, following an
8.7 4.4 21.(~53.5
29.9 12.9
>_-5 38.6 11.7
total 13.7 5.1
2-4
33.0-65.5
38.1 11.1
15
37§-0~!1
51.8 11.1
total 7.0 2.7
2-4
8.0 31.5
12.4 7.5
15
37§ 22 i
19.4 7.2
total
1.6 1.4
2 4
1.0-7.0
1.6 1.4
15
37§ 37"1!
* Standard rosette test performed on 8 animals as described in text, except with variation in incubation temperatures. ++ Percentage of E-rosette forming cells expressed as cells with from 2 to 4, 5 or more, and total with 2 or more attached nSRBC. § Mixtures of bovine peripheral blood lymphocytes and nSRBC were held at this initial incubation temperature for 5 rain. II Lymphocyte nSRBC pellets collected by centrifugation at 200 x g for 5 min were held at this temperature for 24 hr before rosette enumeration.
Mean S.D. Range of total E-rosettes
2-4
22§ 0'I
PBL forming E-rosettes ( ~
Table 2. Influence of incubation temperatures on E-rosette formation by bovine PBL*
3.2 2.4
total
OO
"O
0=
Q
O
Z O ,=
4.4 3.1 2.0 16.0
1.8 1.8
) 5 6.2 4.6
total 9.8 2.3
2 4
6.0-23.0
5.2 3.5
) 5
1.5 hr
15.0 5.1
total 13.1 4.5
2-4
18.0 37.0
12.8 2.9
) 5
4 hr
(/o)°/
25.9 6.5
total
14.1 3.2
2-4
29.0-52.0
27.9 6.3
~>5
24 hr
42.0 6.8
total
* Standard rosette test performed on 8 animals as described in text, except with variation in length of time the bovine lymphocyte- nSRBC pellets were held at 0 C. ++Percentage of E-rosette forming cells expressed as cells with from 2 to 4, 5 or more, and total with 2 or more attached nSRBC.
Mean S.D. Range of total E rosettes
2 4
0 hr
PBL-forming E-rosettes after incubation at 0 C
Table 3. E-rosette formation between bovine PBL and NSRBC incubated at 0 C tbr different times*
Z
N o n - i m m u n e rosette formation of bovine lymphocytes
83
initial incubation at 3 7 C for 5 rain, rosette formation was significantly impaired. Incubation of PBL-nSRBC pellets at 2 2 C for 24 hr supported formation of significantly more rosettes than incubation at 37"C (Table 2).
Effect of centriJ~tgation on E-rosette Jbrmation Centrifugation of PBL-nSRBC mixtures at 200 x g for 5 min enhanced rosette formation as compared to parallel cell mixtures that were not centrifuged. In tests using centrifugation 51.8°o_+ 11.1 of bovine PBL formed rosettes, whereas in tests not using centrifugation there were only 34.6~o_+ 14.2 rosettes, a significant difference (P ~< 0.05).
E[['ect q[" incubating PBL-nSRBC pellets at O C.[br d([ferent times Increasing the length of time that PBL-nSRBC pellets were incubated at 0 'C enhanced E-rosette formation (Table 3). Of the time periods studied, optimum rosette formation occurred at 24 hr. Percentages of lymphocytes with a total of two or more attached nSRBC were significantly higher at 24 hr than for other time periods studied. As the length of incubation time in the ice bath increased, there was, in general, a progressive increase in percentage of lymphocytes with larger numbers of attached nSRBC.
Membrane markers on lymphocytes from bovine fetal thymus tissue The distribution of E-rosette forming lymphocytes and lymphocytes with mIg in thymus tissue from 15 bovine fetuses was studied. All fetal thymuses had high concentrations (92.1'~ _+2.4) of rosette forming cells and low concentrations (2.2°!o_+ 1.4) of cells with mIg. As with bovine peripheral blood mononuclear cell preparations, it was possible using the E-rosette and IFT assays to differentiate from 90.2 to 97.50~ (94.20,'o_+2.1) of cells in bovine fetal thymus preparations into presumptive T and B cells. A high proportion of the rosetteforming cells had five or more attached nSRBC. In fact morulas of nSRBC often formed around bovine fetal thymocytes. Morphologic examination of Wright's stained smears revealed the vast majority (>99?,o) of bovine fetal thymocytes rosetting with nSRBC were lymphocytes.
Ef['ect qf antiglobulin serum on E-rosette formation Antiglobulin serum had no effect on rosette formation by bovine PBL from seven different animals studied. In samples treated with antiglobulin serum 47.9~/o_+9.3 of PBL formed o/ rosettes while in the untreated samples there were 48.7/o _+8.9 rosettes.
Simultaneous detection of E-rosettes and mlg bearing lymphocytes A combined assay for mIg fluorescence and E-rosette formation was conducted on PBL preparations from 10 cattle. Few of the lymphocytes with adherent SRBC were positive for mIg (0.6~,'~_+0.5). In these PBL populations 48.2~/o _+7.6 formed E-rosettes and 24.6~o + 3.1 expressed mlg fluorescence. This study provided further support that the described Erosette and mIg fluorescence methodologies could be used to enumerate presumptive bovine T and B lymphocytes, respectively.
84
HARLAND W. RENSHAW
DISCUSSION This study demonstrates that a certain population of bovine peripheral blood lymphocytes has receptors for SRBC and that these reactive receptor-bearing cells can be detected with a specific procedural technique. Conditions found to be optimal for enumeration of these cells are similar to those reported as optimal for detection of human T cells [9, 13, 14]. Neuraminidase pre-treatment of SRBC enhances E-rosette formation by bovine lymphocytes [2, 3, 7] as it reportedly does for human lymphocytes [15 17]. In contrast to another reported method [3], I chose not to neuraminidase-treat lymphocytes because some cotntroversy exists about whether such treatment actually increases the numbers of T lymphocytes or increases the number of non-T cells that rosette [15]. The available evidence indicates the SRBC receptor on bovine lymphocytes closely resembles the nonspecific erythrocyte-receptor on T cells of a number of other mammalian species. A population of peripheral blood lymphocytes in man [18], nonhuman primates [19, 20], cats [21], dogs [22], pigs [19, 23, 24], horses [25], mice [26], and guinea pigs [9, 19] have receptors for heterologous erythrocytes. It is generally agreed from these and other studies that the erythrocyte-receptor is a useful and accurate T-cell marker. In several species where optimal conditions for E-rosette formation have been carefully documented it is known that the vast majority of thymocytes form E-rosettes. Reports indicate that 98°/~ or more of human thymocytes are capable of forming nonimmune Erosettes with SRBC [27-29] and that 92 99}/0 of guinea pig thymocytes rosette with rabbit erythrocytes [9, 30]. Comparative studies of E-rosette forming cells in bovine PBL and fetal thymocyte preparations reported herein provided convincing evidence of the thymic origin of the bovine rosetting cells. A higher percentage of E-rosettes were found in fetal thymocyte (92.1°/0 _+2.4) than PBL (48.27/0_ 8.4) preparations. Cells with mlg, a marker of B lymphocytes, were found in low concentrations in the bovine fetal thymus (2.2~ _+ 1.4), and in PBL (27.1~ + 2.3) preparations at levels comparable to those previously reported for normal cattle [3, 7, 31 34]. Controversy exists about the comparative efficiency of the different test systems for detection of bovine T lymphocytes. Grewal et al. reported that a mean of 45~o of bovine fetal thymocytes formed E-rosettes using their optimal test conditions [3]. Somewhat higher mean percentages of E-rosette forming thymocytes (61.3~) were reported by Fruchtmann et al. [2]. Higgins and Stack obtained values ranging from 13 to 60.60/0 without lymphocyte storage in FCS at 4~'C for 18 hr before the rosette test [1], while mean values were as high as 66.2% after storage [35]. In each of these studies higher percentages of E-rosette forming thymocytes were expected than were actually found. Incorporation of dextran [4, 8] or dextran and 2 aminoethylisothiouronium bromide (AET) [5, 6] into the test medium reportedly enhances E-rosette formation by bovine PBL and thymocytes. Wardley detected approximately 70~o and 96~ E-rosette forming cells in PBL and thymocyte preparations, respectively [8]. However, others have been unable to obtain comparably high percentages of E-rosettes in thymocyte or PBL preparations regardless of the technology used. The method described by Grewal and Babiuk detected 60-68~ E-rosette forming cells in PBL and 83 84~ in thymocyte preparations [5], whereas, Paul et al. found 38.5-88~ (average 63~) in PBL and 83 90~ in thymocyte preparations [6]. Questions can be raised about the efficiency of these methodologies because of low percentages of E-rosettes in the thymocyte preparations as compared to what should be
Non-immune rosette formation of bovine lymphocytes
85
expected and the high percentages of E-rosettes in the PBL preparations as compared to other reports. The treatments employed may actually increase the number of non-T cells that rosette, rather than increasing numbers of detectable T lymphocytes. Using the E-rosette and mlg assays it was possible in this study to differentiate from 57.5 to 90~o (75.2% + 9.3) of PBL from nine cattle into presumptive T and B cells. Although monocytes also have mlg, the high percentages of lymphocytes in the preparations (mean of 91,°o lymphocytes and 5~o monocytes) suggests that the vast majority of mononuclear cells with mlg in the PBL preparations were B lymphocytes. As in human PBL preparations, T lymphocytes are apparently more numerous in the peripheral blood of cattle than B lymphocytes and there is a population of lymphocytes that cannot be accounted for as Ig-bearing or SRBC-binding lymphocytes. Part of these 'null' cells may be a population oflymphocytes bearing receptors for Fc and C3, but lacking mlg or SRBC receptors. It was shown, in a previous study, that the majority of bovine lymphocytes (87°Jo) having Fc receptors also possessed mlg, and the percentages of cells having mlg and Fc receptors closely paralleled each other in studies of 24 clinically normal cattle [7]. Further when a combined E- and EA-rosette assay for simultaneously detecting presumptive T and B cells was conducted, sub-populations of cells were found with both receptors (1.25~o) and no receptors (21.2~,). In the present study only 0.6~o+0.5 of lymphocytes with adherent SRBC also possessed mlg. An E-rosette forming cell was defined as a lymphocyte with as least two adherent SRBC, whereas, others have defined it as a cell with more adherent SRBC. While this tends to increase the percentage of E-rosettes counted, the data make it apparent that the majority of these cells have five or more adherent nSRBC. With most species studied thus far, optimal rosette formation is very much dependent upon procedural techniques. Modifications of the E-rosette assay reported herein may be found which will further improve the accuracy of the test. Ongoing investigations to enhance the precision of the T- and B-cell enumerations are directed at development of a modification, which would allow for specific identification of monocytes in PBL preparations. With the development of reliable techniques for their enumeration, major advances should be forthcoming in understanding the role T and B lymphocytes play in immune expression in both normal and diseased cattle.
REFERENCES 1. Higgins,D. A. and Stack, M. J., Bovinelymphocytes:recognitionof cells formingspontaneous (E) rosettes, Clin. exp. Immunol. 27, 348 354 (1977). 2. Fruchtmann, R., Uhlenbruck, G., Schmid, D. V. and Cwik, S., Zur charakterisierung yon T- und Blymphozyten beim rind, Zentbl. Vet Med. B. 24, 486-496 (1977). 3. Grewal, A. S., Rous, B. T. and Babiuk, L. A., Erythrocyterosettes a marker for bovineT cells, Can. J. comp. Med. 40, 298-305 (1976). 4. Binns, R. M., Sheep erythrocyte rosettes in pigs, sheep, cattle and goats demonstrated in the presence of dextran, J. lmmunol. Method~ 21, 197-210 (1978). 5. Grewal, A. S. and Babiuk, L. A., BovineT-lymphocytes an improved technique of E-rosette formation, J. lmmunol. Methods 24, 355-361 (1978). 6. Paul, P. S., Senogles, D. R., Muscoplat, C. C. and Johnson, D. W., Enumeration of T cells, B cells and monocytes in peripheral blood of normal and lymphocyticcattle, Clin. exp. Immunol. 35, 306-316 (1979). 7. Reeves,J. H. and Renshaw, H. W., Surfacemembrane markers on bovine peripheral blood lymphocytes,Am. J. vet. Res. 39, 917-923 (1977). 8. Wardley, R., An improved E-rosette technique for cattle, Br. vet. J. 133, 432 434 (1977). 9. Wilson, A. B. and Coombs, R. R. A., Rosette-formation between guinea pig lymphoid cells and rabbit erythrocytes--a possible T-cell marker, Int. Archs Allergy appl. Immunol. 44, 544-552 (1973).
86
tIARLAND W. RI:NSItAW
10. Herbert, W. J., Passive haemagglutination. In Handbook O/' Evperimental Immunology, Weir, D. W. (ed.), Blackwell Scientific, Oxford pp. 720 744 (1967). 11. B6yum, A., Isolation of mononuclear cells and granulocytes from human blood, Stand. J. olin. Lab. Inves't. Suppl. 97, 77 89 (1968). 12. Hummeler, K., Harris, T. N., Tomassini, N., Hechtel, M. and Farber, M. B., Electron microscopic observations on antibody-producing cells in lymph and blood, J. exp. Med. 12.4, 255 262 (1966). 13. Aiuti, F., Cerottini, J.-C., Coombs, R. R. A., Cooper, M., Dickler, H. B., Froland, S. S., Fudenberg, H. H. Greaves, M. F., Gray, H. M., Kunkel, H. G., Natvig, J. B., Preud'Homme, J.-L., Rabellino, Ritts, R. E., Rowe, D. S., Seligmann, M., Siegal, F. P., Stjernsward, L, Terry, W. D. and Wybran, J., Identification, enumeration, and isolation of B and T lymphocytes from human peripheral blood, Stand. J. Immunol. 3, 521-532 (1974). 14. Weiner, M. S., Bianco, C. and Nussenzweig, V., Enhanced binding of neuraminidase-treated sheep erythrocytes to human T lymphocytes, Blood 42, 939-946 (1973). 15. Bentwich, Z., Douglas, S. D., Skutelsky, E. and Kunkel, H. G., Sheep red cell binding to human lymphocytes treated with neuraminidase: enhancement of T cell binding and identification of a subpopulation of B cells, J. exp. Med. 137, 1532 1537 (1973). 16. Galili, U. and Schlesinger, M., The formation of stable E rosettes after neuraminidase treatment of either human peripheral blood lymphocytes or of sheep red blood cells, J. lmmunol. 112, 1628 1634 (1974). 17. Han, T. and Minowada, J., Enhanced E- and EAC-rosette formation by neuraminidase, J. Immunol. Methods 12, 253 260 (1976). 18. Hallberg, T., Human lymphocyte subpopulations, Acta path. microbiol, stand. B. Suppl. 250, 1 54 (1975). 19. Johansen, K. S., Johansen, T. S. and Talmage, D. W., T cell rosette formation in primates, pigs, and guinea pigs. The influence of immunosuppressive agents, J. Allergy olin. lmmunol. 54, 86 93 (1974). 20. Terrell, T. G., Holmberg, C. A. and Osburn, B. I., Immunologic surface markers on nonhuman primate lymphocytes, Am. J. vet. Re.s. 38, 503 507 (1977). 21. Mac Key, L. J., Distribution o f t and B cells in thymus, blood and lymph nodes of the cat, Res. vet. Sci. 22,225 228 (1977). 22. Bowles, C. A., White, G. S. and Lucas, D., Rosette formation by canine peripheral blood lymphocytes. J. hnmunol. 114, 399 402 (1975). 23. Escajadillo, C. and Binns, R. M., Rosette formation with sheep erythrocytes--A possible T-cell marker in the pig, Int. Archs Allergy appl. Immunol. 48, 261 275 (1975). 24. Shimizu, M., Pan, I. C. and Hess, W. R., T and B lymphocytes in porcine blood, Am. J. vet. Res. 37, 309 317 (1976). 25. Tart, M. J., Olsen, R. G , Krakowka, G. S., Cockerell, G. L. and Gabel, A. A., Erythrocyte rosette formation of equine peripheral blood lymphocytes, Am. J. vet. Res. 38, 1775 1779 (1977). 26. Charrier6, J., Dardenne, M. and Bach, J. F., Antigen recognition by T lymphocytes. IV. Differences in antigen-binding characteristics of T- and B-RFC: a cause for variations in the evaluation of T-RFC, Cell. lmmunol. 9, 32-44 (1973). 27. Jondal, M., Holm, G. and Wigzell, H., Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming non-immune rosettes with sheep erythrocytes, J. exp. Med. 136, 2(t7 215 (1972). 28. Lay, W. H., Mendes, N. F., Bianco, C. and Nussenzweig, V., Binding of sheep red blood cells to a large population of human lymphocytes, Nature, Lond 230, 531 532 (1971). 29. Silveira, N. P. A., Mendes, N, F. and Tolnai, M. E. A., Tissue localization of two populations of human lymphocytes distinguished by membrane receptors, J. lmmunol. 108, 1456 1460 (1972). 30. Stadecker, M. J., Bishop, G. and Wortis, H. H., Rosette formation by guinea pig thymocytes and thymus derived lymphocytes with rabbit red blood cells, J. lmmunol. 111, 1834 1837 (1973). 31. Kenyon, S. J. and Piper, C. E., Cellular basis of persistent lymphocytosis in cattle infected with bovine leukemia virus, Inject. lmmun. 16, 891 897 (1977). 32. Muscoplat, C. C., Johnson, D. W., Pomeroy, K. A., Olson, J. M., Larson, V. L., Stevens, J. B. and Sorensen, D. K., Lymphocyte surface immunoglobulin : frequency in normal and lymphocytic cattle, Am. J. vet. Res. 35, 593 595 (1974). 33. Muscoplat, C. C., Johnson, D. W., Pomeroy, K. A., Olson, J. M., Larson, V. L. Stevens, J. B. and Sorensen, D. K. Lymphocyte subpopulations and immunodeficiency in calves with acute lymphocytic leukemia, Am. J. vet. Res. 35, 1571 1573 (1974). 34. Muscoplat, C. C., Johnson, D. W. and Teuscber, E., Surface immunoglobulin of circulating lymphocytes in chronic bovine diarrhea: abnormalities in cell populations and cell function, Am. J. vet. Res. 34, 1101 1104 (1973). 35. Higgins, D. A. and Stack, M. J., Bovine lymphocytes: enhanced E-rosette formation after storage or gradient centrifugation, Clin. exp. lmmunol. 29, 304 310 (1977). 36. Pang, G. T. M., Baguley, D. M. and Wilson, J. D., Spontaneous rosettes as a T-lymphocyte marker: a modified method giving consistent results. SRBC rosettes, J, lmmunol. Methods' 4, 41 46 (1974).
0147 9571/81/010075-12 $02.00/0 ~ 1981 PergamonPressLtd.
Printed in Great Britain.
N O N - I M M U N E E R Y T H R O C Y T E ROSETTE F O R M A T I O N OF BOVINE P E R I P H E R A L BLOOD A N D THYMUS LYMPHOCYTES HARLAND W . RENSHAW Department of Veterinary Microbiology and Parasitology, Texas Agricultural Experiment Station, College of Veterinary Medicine, Texas A&M Universit L College Station, TX 7784K U.S.A. Abstract--An E-rosetting reaction is described which gave 92.1°~_+ 2.4 (mean_+ S.D.) E-rosettes with bovine fetal thymocytes and 48.2~o + 8.4 with with bovine peripheral blood leukocyte (PBL) preparations. Both culture conditions and culture medium were critical factors in obtaining maximal and reproducible E-rosette numbers. Optimum rosette formation occurred when bovine PBL and neuraminidase treated sheep erythrocytes (nSRBC) were reacted in L-15 culture medium supplemented with 10°~, fetal calf serum (FCS). Other media including 100~o FCS, M E M with 10~o FCS, and RPMI-1640 with 100/oFCS were less satisfactory. Cultural conditions found to be optimal for enumeration of bovine E-rosettes are similar to those reported as optimal for detection of human T cells. The specificity of rosette formation by bovine thymus derived (T) lymphocytes was shown by demonstration of (1) rosettes and surface membrane immunoglobulins (mIg) on different cells in PBL, (2) rosette formation by the majority of fetal thymocytes, and (3) no inhibition of rosette formation by anti-immunoglobulin serum. Using the E-rosette and mIg assays for presumptive bovine T and B lymphocytes, respectively, it was possible to differentiate from 57.5 to 900 (75.2°0 _+9.3) of cells in bovine PBL preparations, and from 90.2 to 97.50/o (94.2°% _+2.1) of cells in bovine fetal thymocyte preparations into T and B cells. Key words: Non-immune erythrocyte rosettes, E-rosettes, T cells, T lymphocytes, bovine T and B
lymphocytes LA FORMATION LYMPHOCYTES
DE ROSETTES DE BOVIN
NON
IMMUNES
ISSUS DU
DE
SANG
PERIPHERIQUES OU DU THYMUS R o s u m ~ - La reaction de rosettes E decrite donne 92,1"~_+ 2,4 de rosettes avec les lymphocytes du thymus et 48,2%o_+8,4 avec les preparations leucocytaires issues du sang peripherique (PBL). Les conditions et le milieu de culture sont des facteurs importants pour l'obtention d'un hombre maximum de rosettes E et la reproductibilite de la reaction. La quantit6 optimale de rosettes a 6re obtenue quand la PBL bovine et des ~rythrocytes de mouton impregnes de neuraminidase (nSRBC) ont 6te places dans un milieu de culture L-15 additionne de 10'~,ide serum foetal de veau (FSC). D'autres milieux, dont le FSC seul, le MEM avec 10'~,~FCS, et le RPMI-1640 avec 10!i~ FSC laissaient ~i d6sirer. Les conditions de culture qui ont ete decrites comme optimales pour l'obtention des rosettes E bovines, sont semblables fi celles rapportees comme optimales pour les cellules T humaines. Trois points ont permis de d6montrer la spbcificit6 des cellules du thymus formant des rosettes: II existe des immunoglobulines M sur la membrane de quelques cellules issues du sang peripherique. (2) La majorite des thymocytes foetaux forment des rosettes. (3) la formation des rosettes n'est pas inhibee par un s~rum antiimmunoglobuline. En utilisant les rosettes E el les dosages des immunoglobulines M pour identifier, respectivement, les lymphocytes T et les lymphocytes B, nous avons pu caracteriser 57,5 fi 90')o (75,27 _+9,3) des cellules du sang p6ripherique et 90,2 fi 97, 5"% (94,2~o _+2,1) des cellules thymiques en lymphocytes T ou B. Mots-clef~: Rosettes erythrocytes non-immunes, rosettes E, cellules T~ lymphocytes T, lymphocytes bovins T et B INTRODUCTION
It is now generally accepted that in all mammalian species there are two distinct classes of lymphocytes, each with different functional activities and cell surface membrane 75
76
HARLAND W. RENSHAW
receptors. The T lymphocytes of many species have been shown to form non-immune erythrocyte (E) rosettes with heterologous erythrocytes, and the B lymphocytes to bind fluorescein-labeled antiserum to immunoglobulins of the species involved. Several groups have recognized E-rosette formation with bovine lymphocytes incubated under appropriate conditions with suitably prepared sheep red blood cells (SRBC). Although each group concluded that E-rosettes could be used as a marker of bovine T cells, there were significant differences in the percentage of E-rosette forming lymphocytes reportedly found in peripheral blood and thymus. Higgins and Stack suggested that only a limited population of bovine T cells, possibly immature bovine T cells, form E rosettes and that this sub-population is either absent or present in low percentages in peripheral blood of calves and adults [1]. In several studies it has been speculated that even under the experimental conditions found to be optimum that not all bovine T cells formed E rosettes [1 3]. Studies of E-rosette forming cells in other species suggest that slight variations in procedural techniques can account for dramatic differences in percentages of E-rosette forming lymphocytes found in the peripheral blood or organs of normal subjects. Different treatments of SRBC or additives to the rosetting medium have been used to enhance rosette formation by presumptive bovine T lymphocytes [3 8]. Considerable confusion exists about the reliability of the various different methods described for demonstrating presumptive bovine T lymphocytes because investigators have not always adequately demonstrated the specificity of their E-rosetting reaction. In some cases, the specificity of the E-rosetting reaction has been proposed as adequate if E-rosette forming cells were found in percentages that closely approximated what was thought to be the expected distribution of T cells in bovine peripheral blood and thymus lymphocyte preparations. The expected distribution being extrapolated from previously published data about T-cell distribution in humans and laboratory animals. If an E-rosette forming reaction is to be considered as a T-cell marker for cattle the rosette-forming reaction should: (1) occur with the majority of thymus cells and with a large enough percentage of peripheral blood lymphocytes as to exclude reaction by specific immunoglobulin receptors, (2) occur with a cell population separate from that bearing detectable immunoglobulin determinants, and (3) not be inhibited by anti-immunoglobulin serum [9]. To date none of the described methods for detecting presumptive bovine T lymphocytes has been shown to completely fulfill each of these requirements. In a previous study, techniques were evaluated for identifying sub-populations of bovine peripheral blood lymphocytes [7]. Presumptive bovine T cells were identified in peripheral blood lymphocyte preparations using conditions similar to those reported as optimal for human cells. Evidence will be presented in this report that (1) this E-rosette forming reaction fulfills each of the above indicated requirements and is therefore a reliable marker for bovine T lymphocytes, and (2) both culture conditions and media are critical factors in the non-immune E-rosette technique with bovine lymphocytes.
MATERIALS AND METHODS Lymphocyte sources
Heparinized blood samples (15 U/ml) were collected by jugular venipuncture from Holstein Friesian or cross-bred beef cattle of different ages. All animals were fed a
Non-immunerosetteformationof bovine lymphocytes
77
standard ration and were clinically normal. Bovine fetal thymus tissue was obtained at a local abattoir. Normal appearing fetuses, 7-9 months of estimated gestational age, were obtained from beef type cattle passed for human consumption by government inspection.
Sheep erythrocytes (SRBC) Venous blood from clinically healthy sheep was collected into sterile Alsever's solution pH 6.1 [10]. Collected sheep blood was stored as a 50~ suspension in Alsever's solution at 4'C, and used in the E-rosette assay between 1 and 3 weeks afer collection. Before use, SRBC were washed twice in sterile physiologic saline (PS), recovered between washings by centrifugation at 400 x g for 10 min at 22'~C, and resuspended to 0.5% in the appropriate medium.
Neuraminidase treatment of SRBC The SRBC used in the E-rosette assay were prepared for use as described above or after neuraminidase treatment with Vibrio cholerae filtrate (Sigma Chemical Co., St Louis, MO) that was reconstituted (0.05 U/ml) with PS. A mixture of 1 vol of packed washed SRBC and 5 vol of reconstituted filtrate were incubated at 37~C for 60 min. The treated cells were washed twice in the appropriate medium.
Media and sera The same lot of fetal calf serum (FCS) was used throughout this study (lot 086312, Grand Island Biological Co., Grand Island, NY). The FCS was inactivated at 56°C for 30min and absorbed at 37~:C for l hr against an equal volume of packed SRBC. Fluorescein-conjugated (FITC) rabbit antiserum prepared against the 7S globulin fraction of bovine serum (RaBIg) was purchased (Miles Laboratories, Inc., Elkhart, IN). Several commercially available media (Grand Island Biological Co., Grand Island, NY), including Leibovitz medium (L-15), RPMI 1640, and Eagle's minimal essential medium (MEM), all at pH 7.4, were compared for their capacity to support E-rosette formation.
Peripheral blood lymphocyte isolation Bovine peripheral blood lymphocytes (PBL were isolated using a modified version of the method of B6yum [11]. Heparinized blood (15 ml) was mixed with an equal volume of L-15, or another appropriate medium containing 15 U of heparin/ml in a 50-ml conicalbottom plastic screw-top centrifuge tube. Sucrose copolymer/sodium diatrizoate (24 parts of 9~o w/v aqueous solution of Ficoll 400, Pharmacia Fine Chemicals, Uppsala, Sweden, 10 parts of 33.9% w/v aqueous solution of Hypaque, Winthrop Laboratories, NY, with specific gravity adjusted to 1.077) solution (20 ml) was layered beneath the blood-media mixture, and tubes were centrifuged at 400 x g for 40 min at 22°C. The mononuclear cell layer at the gradient interface was collected, contaminating erythrocytes were removed by lysis with 3 vol of 0.87~o NH4C1 [2], and the leukocytes recovered by centrifugation at 400 x g for 10 min at 22"C. The cells were washed twice in PS with heparin (15 U/ml), and adjusted to 15 x 106 lymphocytes/ml. Differential leukocyte counts were performed on Wright's stained smears. The cells were then washed two more times in the appropriate test medium.
78
HARLAND W. RENSHAW
Cell suspensions free of erythrocytes contained approximately 91~o lymphocytes O . .l . .9. °Jo/ -+ 5.8" range 82-99%) and 5 ~ monocytes (5.1 o~o -+ 4.4; range 1-15%). Greater than 95",~i of the cell population was capable of excluding 0.1% trypan blue in all experiments.
Thymocyte isolation Bovine fetal thymus tissue was transported on ice in phosphate-buffered saline (pH 7.4). The thymic tissue was dissected free of connective tissue and blood vessels, minced, and gently squeezed with a rubber policeman through a 120 mesh stainless sieve into PS with heparin (15 U/ml). The suspension was centrifuged at 4 0 0 x g for 10 min at 2 2 C , then resuspended in 3 vol of 0,87°,o NHaC1 to remove contaminating erythrocytes. The thymocytes were collected by centrifugation, washed twice in L- 15 and resuspended in L- 15 with 100~i FCS at 1 5 x 1 0 ~' thymocytes/ml. The final cell suspension contained approximately 97°;; lymphocytes (97.4°o_+2.4; range 93 100'~'o) and greater than 96°~i of the cell population was capable of excluding trypan blue.
Bovine E-rosette test A suspension of 2.25 x 106 PBL or thymocytes in 0.15 ml of L- 15 or another appropriate medium with lO',!Jo FCS was combined with 0.3 ml of a 0.5°J~i suspension of either neuraminidase treated (nSRBC) or untreated SRBC in L-15 or another appropriate medium with 10~o FCS. The cell mixture was incubated for 5 min in a 37~'C water bath, centrifuged at 200 x g for 5 rain at 2 2 C , and then incubated in an ice water bath (OC) for 24 hr. The cell pellet was resuspended and a drop of 0.21!,~itrypan blue added. An aliquot of the cell suspension was then added to a pre-chilled (4:'C) Neubauer counting chamber and t h e percentage of viable leukocytes binding two or more SRBC determined microscopically at 440 x . At least 200 mononuclear cells were examined for E-rosette formation for each sample. Cell counts were made between 1 and 10 min after placing the cell suspension in the counting chamber. The percentage of E-rosette forming cells with two or more SRBC attached was determined.
lmmunofluorescence test (IFT)./'or immunoglobulin-bearing l)'mpho~3,tes A suspension of 3 x 106 PBL or thymocytes in 0.2 ml L-15 was mixed with 0.2 ml o f a 1 : 10 dilution of FITC-labelled RaBIg, incubated at 4°C for 30 min with occasional gentle shaking, washed three times with L-15 at 4°C, and recovered between each wash by centrifugation at 400 x g for 5 min. The cell pellet was resuspended in several drops of glycerol: PBS (1 : 1) mounting medium and examined by fluorescence microscopy at 440 x using a fluorescence microscope equipped with mercury vapor lamp illumination (Carl Zeiss, NY). Four hundred cells were examined under both incandescent light and ultraviolet (u.v.). Cell membrane bound immunoglobulin (mIg) usually appeared as an homogeneous speckled distribution over the entire cell or as 'caps'. For simultaneous detection of E-rosettes and mIg-bearing lymphocytes the bovine cell preparations were reacted with FITC-labelled RaBIg as described above. Subsequently, to allow formation of E-rosettes, 0.1 ml of labelled leukocytes was combined with 0.2 ml of a 0.5% suspension of nSRBC in L-15 with 10% FCS. The labelled bovine leukocyte-nSRBC mixture was incubated for 5 rain at 37°C, pelleted by centrifugation at 200 x g at 22°C for 5 min, and
2.5
5.3
2.0 2.0 3.0 6.5 5.5 8.0 5.0 8.5 7.0
2-4
2.0-25.0
6.5
9.3
0.0 1.0 5.0 18.0 8.0 9.0 11.5 16.5 14.5
/> 5
8.7
14.6
2.0 3.0 8.0 24.5 13.5 17.0 16.5 25.0 21.5
total
L - 1 5 + 10% FCS with SRBC§
2.1
11.9
11.5 12.5 11.5 9.0 16.5 12.0 10.0 12.5 12.0
2 4
35.5 61.0
9.3
36.3
24.0 27.0 33.0 49.5 27.0 39.0 39.5 38.5 49.0
/> 5
8.4
48.2
35.5 39.5 44.5 58.5 43.5 51.0 49.5 51.0 61.0
total
L - 1 5 + 10% FCS with nSRBCII
1.7
5.0
5.0 3.0 4.0 6.5 7.5 4.5 0.5 2.5 5.5
2 4
20.0 55.0
11.1
33.2
21.0 17.0 28.0 48.5 34.0 40.5 30.0 30.5 49.0
~>5
12.0
38.2
26.0 20.0 32.0 55.0 41.5 45.0 36.0 32.5 54.5
total
100% FCS with nSRBC
2.8
7.4
6.0 9.0 4.0 9.0 7.5 11.0 7.0 3.0 10.5
2-4
7.5-43.5
11.0
14.0
3.0 2.0 3.5 27.0 8.5 20.0 15.5 13.5 33.0
/> 5
12.8
21.4
9.0 11.0 7.5 36.0 16.0 31.0 22.5 16.5 43.5
total
R P M I + 10% FCS with n S R B C
3.6
7.3
4.0 3.0 3.5 8.0 8.0 8.5 11.0 6.0 14.0
2 4
8.0-39.0
8.8
17.7
17.0 16.0 4.5 31.0 11.0 30.5 11.0 18.5 20.0
>/5
10.4
25.0
21.0 19.0 8.0 39.0 19.0 39.0 22.0 24.0 34.0
total
M E M + 10% FCS with n S R B C
* Standard rosette test performed as described in text, except with variation of media. L-15 = Leibovitz medium, FSC = fetal calf serum: RPMI = RPMI-1640; M E M = Eagle's minimal essential medium. Percentage of E-rosette forming cells expressed as cells with from 2 to 4, 5 or more, and total with 2 or more attached sheep erythrocytes. ++ mIg-Bearing cells are those cells showing m e m b r a n e fluorescence after treatment with fluorescein-conjugated rabbit anti-bovine immunoglobulin G; percentage of 400 cells. § SRBC = untreated sheep erythrocytes. II nSRBC = neuraminidase-treated sheep erythrocytes.
22.0-29.2
2.3
Range (mIg or total Erosettes)
27.1
1' 2 3 4 5 6 7 8 9
S.D.
22.0 28.5 27.5 25.0 29.2 27.0 28.5 26.8 29.0
Animal number
Mean
Lymphocytes with mlg~ (%)
PBL forming E-rosettes (%)*
Table 1. E-rosette formation by bovine PBL after incubating lymphocyte-SRBC pellets in different media
80
HARLAND W. RENSHAW
incubated for 24 hr at 0°C. The percentages of leukocytes with the different surface markers were determined for at least 200 mononuclear cells in a pre-chilled counting chamber under both incandescent light and u.v.
Influence of antiglobulin serum on E-rosette formation PBL were treated for 1 hr at 4°C with a 1/10 dilution of heat inactivated rabbit antibovine IgG containing 3.1 mg antibody protein per ml antiserum (Miles Laboratores Inc., Elkhart, IN). This concentration of antibody prevented immunofluorescent staining of immunoglobulin-bearing bovine lymphocytes. Control tubes were treated with heat inactivated normal rabbit serum at the same dilution. Cells were washed three times in L-15 with 10% FCS to remove excess serum before rosetting with nSRBC. RESULTS
Effect of incubating PBL-SRBC mixtures in different media Several different media were compared for their capacity to support formation of spontaneous, direct E-rosettes between bovine PBL and nSRBC. Bovine PBL and nSRBC were incubated for 5 min at 37°C, centrifuged at 200 × g for 5 min at 22°C, and incubated on ice (0c'C) for 2 4 h r with the following media: L - 1 5 + 1 0 ~ FCS, R P M I + I 0 ~ FCS, M E M + 10~ FCS, and 100~o FCS. Additionally, bovine PBL were mixed with untreated SRBC in L-15 + 10~ FCS. Parallel test results of E-rosette formation with bovine PBL in these various media are presented in Table 1. Optimum rosette formation occurred when neuraminidase treated SRBC were reacted with PBL in L-15 supplemented with 10~ FCS. Lymphocytes reacting in the medium gave the highest mean percentage of E-rosettes. For each animal tested the percentage of E-rosettes formed was highest with this medium. With the other media tested, the next highest percentage of rosettes formed when bovine PBL and nSRBC were incubated in 100% FCS. Clumping of lymphocytes occurred when some cell samples were incubated in 100~ FCS. This made interpretation of the E-rosette assay difficult when this medium was used. RPMI-1640+10~o FCS or M E M + 1 0 ~ FCS yielded the lowest number of rosette-forming cells. Treatment of SRBC with neuraminidase significantly enhanced rosette formation when bovine PBL-SRBC mixtures were incubated in L-15 + 10~ FCS (Table 1). In paralled with the rosette study, PBL were examined for mlg. Approximately 27~ of the PBL from the 9 clinically normal Holstein-Friesian cattle were positive for mlg (Table 1). Using the direct fluorescence test for detection of cells with mlg and the E-rosette test with L-15+ 10~ FCS and nSRBC it was possible to differentiate for 57.7 to 90~o (75.2~+9.3) of cells in bovine PBL preparations into presumptive T and B cells. Greater than 99~, of the bovine cells rosetting with nSRBC were identified as lymphocytes when Wright's stained smears were examined.
Effect of incubation temperature on E-rosette formation Bovine PBL and nSRBC mixtures in L - 1 5 + 10% FCS were incubated at either 37 or 2 2 C for 5 rain before centrifugation and incubation for 24 hr at WC. In all cases the percentages of E-rosette forming cells was higher at 37 than at 22°C (Table 2). If the PBLnSRBC pellets were held at either 22 or 37~C for 24 hr, rather than at 0"C, following an
8.7 4.4 21.(~53.5
29.9 12.9
>_-5 38.6 11.7
total 13.7 5.1
2-4
33.0-65.5
38.1 11.1
15
37§-0~!1
51.8 11.1
total 7.0 2.7
2-4
8.0 31.5
12.4 7.5
15
37§ 22 i
19.4 7.2
total
1.6 1.4
2 4
1.0-7.0
1.6 1.4
15
37§ 37"1!
* Standard rosette test performed on 8 animals as described in text, except with variation in incubation temperatures. ++ Percentage of E-rosette forming cells expressed as cells with from 2 to 4, 5 or more, and total with 2 or more attached nSRBC. § Mixtures of bovine peripheral blood lymphocytes and nSRBC were held at this initial incubation temperature for 5 rain. II Lymphocyte nSRBC pellets collected by centrifugation at 200 x g for 5 min were held at this temperature for 24 hr before rosette enumeration.
Mean S.D. Range of total E-rosettes
2-4
22§ 0'I
PBL forming E-rosettes ( ~
Table 2. Influence of incubation temperatures on E-rosette formation by bovine PBL*
3.2 2.4
total
OO
"O
0=
Q
O
Z O ,=
4.4 3.1 2.0 16.0
1.8 1.8
) 5 6.2 4.6
total 9.8 2.3
2 4
6.0-23.0
5.2 3.5
) 5
1.5 hr
15.0 5.1
total 13.1 4.5
2-4
18.0 37.0
12.8 2.9
) 5
4 hr
(/o)°/
25.9 6.5
total
14.1 3.2
2-4
29.0-52.0
27.9 6.3
~>5
24 hr
42.0 6.8
total
* Standard rosette test performed on 8 animals as described in text, except with variation in length of time the bovine lymphocyte- nSRBC pellets were held at 0 C. ++Percentage of E-rosette forming cells expressed as cells with from 2 to 4, 5 or more, and total with 2 or more attached nSRBC.
Mean S.D. Range of total E rosettes
2 4
0 hr
PBL-forming E-rosettes after incubation at 0 C
Table 3. E-rosette formation between bovine PBL and NSRBC incubated at 0 C tbr different times*
Z
N o n - i m m u n e rosette formation of bovine lymphocytes
83
initial incubation at 3 7 C for 5 rain, rosette formation was significantly impaired. Incubation of PBL-nSRBC pellets at 2 2 C for 24 hr supported formation of significantly more rosettes than incubation at 37"C (Table 2).
Effect of centriJ~tgation on E-rosette Jbrmation Centrifugation of PBL-nSRBC mixtures at 200 x g for 5 min enhanced rosette formation as compared to parallel cell mixtures that were not centrifuged. In tests using centrifugation 51.8°o_+ 11.1 of bovine PBL formed rosettes, whereas in tests not using centrifugation there were only 34.6~o_+ 14.2 rosettes, a significant difference (P ~< 0.05).
E[['ect q[" incubating PBL-nSRBC pellets at O C.[br d([ferent times Increasing the length of time that PBL-nSRBC pellets were incubated at 0 'C enhanced E-rosette formation (Table 3). Of the time periods studied, optimum rosette formation occurred at 24 hr. Percentages of lymphocytes with a total of two or more attached nSRBC were significantly higher at 24 hr than for other time periods studied. As the length of incubation time in the ice bath increased, there was, in general, a progressive increase in percentage of lymphocytes with larger numbers of attached nSRBC.
Membrane markers on lymphocytes from bovine fetal thymus tissue The distribution of E-rosette forming lymphocytes and lymphocytes with mIg in thymus tissue from 15 bovine fetuses was studied. All fetal thymuses had high concentrations (92.1'~ _+2.4) of rosette forming cells and low concentrations (2.2°!o_+ 1.4) of cells with mIg. As with bovine peripheral blood mononuclear cell preparations, it was possible using the E-rosette and IFT assays to differentiate from 90.2 to 97.50~ (94.20,'o_+2.1) of cells in bovine fetal thymus preparations into presumptive T and B cells. A high proportion of the rosetteforming cells had five or more attached nSRBC. In fact morulas of nSRBC often formed around bovine fetal thymocytes. Morphologic examination of Wright's stained smears revealed the vast majority (>99?,o) of bovine fetal thymocytes rosetting with nSRBC were lymphocytes.
Ef['ect qf antiglobulin serum on E-rosette formation Antiglobulin serum had no effect on rosette formation by bovine PBL from seven different animals studied. In samples treated with antiglobulin serum 47.9~/o_+9.3 of PBL formed o/ rosettes while in the untreated samples there were 48.7/o _+8.9 rosettes.
Simultaneous detection of E-rosettes and mlg bearing lymphocytes A combined assay for mIg fluorescence and E-rosette formation was conducted on PBL preparations from 10 cattle. Few of the lymphocytes with adherent SRBC were positive for mIg (0.6~,'~_+0.5). In these PBL populations 48.2~/o _+7.6 formed E-rosettes and 24.6~o + 3.1 expressed mlg fluorescence. This study provided further support that the described Erosette and mIg fluorescence methodologies could be used to enumerate presumptive bovine T and B lymphocytes, respectively.
84
HARLAND W. RENSHAW
DISCUSSION This study demonstrates that a certain population of bovine peripheral blood lymphocytes has receptors for SRBC and that these reactive receptor-bearing cells can be detected with a specific procedural technique. Conditions found to be optimal for enumeration of these cells are similar to those reported as optimal for detection of human T cells [9, 13, 14]. Neuraminidase pre-treatment of SRBC enhances E-rosette formation by bovine lymphocytes [2, 3, 7] as it reportedly does for human lymphocytes [15 17]. In contrast to another reported method [3], I chose not to neuraminidase-treat lymphocytes because some cotntroversy exists about whether such treatment actually increases the numbers of T lymphocytes or increases the number of non-T cells that rosette [15]. The available evidence indicates the SRBC receptor on bovine lymphocytes closely resembles the nonspecific erythrocyte-receptor on T cells of a number of other mammalian species. A population of peripheral blood lymphocytes in man [18], nonhuman primates [19, 20], cats [21], dogs [22], pigs [19, 23, 24], horses [25], mice [26], and guinea pigs [9, 19] have receptors for heterologous erythrocytes. It is generally agreed from these and other studies that the erythrocyte-receptor is a useful and accurate T-cell marker. In several species where optimal conditions for E-rosette formation have been carefully documented it is known that the vast majority of thymocytes form E-rosettes. Reports indicate that 98°/~ or more of human thymocytes are capable of forming nonimmune Erosettes with SRBC [27-29] and that 92 99}/0 of guinea pig thymocytes rosette with rabbit erythrocytes [9, 30]. Comparative studies of E-rosette forming cells in bovine PBL and fetal thymocyte preparations reported herein provided convincing evidence of the thymic origin of the bovine rosetting cells. A higher percentage of E-rosettes were found in fetal thymocyte (92.1°/0 _+2.4) than PBL (48.27/0_ 8.4) preparations. Cells with mlg, a marker of B lymphocytes, were found in low concentrations in the bovine fetal thymus (2.2~ _+ 1.4), and in PBL (27.1~ + 2.3) preparations at levels comparable to those previously reported for normal cattle [3, 7, 31 34]. Controversy exists about the comparative efficiency of the different test systems for detection of bovine T lymphocytes. Grewal et al. reported that a mean of 45~o of bovine fetal thymocytes formed E-rosettes using their optimal test conditions [3]. Somewhat higher mean percentages of E-rosette forming thymocytes (61.3~) were reported by Fruchtmann et al. [2]. Higgins and Stack obtained values ranging from 13 to 60.60/0 without lymphocyte storage in FCS at 4~'C for 18 hr before the rosette test [1], while mean values were as high as 66.2% after storage [35]. In each of these studies higher percentages of E-rosette forming thymocytes were expected than were actually found. Incorporation of dextran [4, 8] or dextran and 2 aminoethylisothiouronium bromide (AET) [5, 6] into the test medium reportedly enhances E-rosette formation by bovine PBL and thymocytes. Wardley detected approximately 70~o and 96~ E-rosette forming cells in PBL and thymocyte preparations, respectively [8]. However, others have been unable to obtain comparably high percentages of E-rosettes in thymocyte or PBL preparations regardless of the technology used. The method described by Grewal and Babiuk detected 60-68~ E-rosette forming cells in PBL and 83 84~ in thymocyte preparations [5], whereas, Paul et al. found 38.5-88~ (average 63~) in PBL and 83 90~ in thymocyte preparations [6]. Questions can be raised about the efficiency of these methodologies because of low percentages of E-rosettes in the thymocyte preparations as compared to what should be
Non-immune rosette formation of bovine lymphocytes
85
expected and the high percentages of E-rosettes in the PBL preparations as compared to other reports. The treatments employed may actually increase the number of non-T cells that rosette, rather than increasing numbers of detectable T lymphocytes. Using the E-rosette and mlg assays it was possible in this study to differentiate from 57.5 to 90~o (75.2% + 9.3) of PBL from nine cattle into presumptive T and B cells. Although monocytes also have mlg, the high percentages of lymphocytes in the preparations (mean of 91,°o lymphocytes and 5~o monocytes) suggests that the vast majority of mononuclear cells with mlg in the PBL preparations were B lymphocytes. As in human PBL preparations, T lymphocytes are apparently more numerous in the peripheral blood of cattle than B lymphocytes and there is a population of lymphocytes that cannot be accounted for as Ig-bearing or SRBC-binding lymphocytes. Part of these 'null' cells may be a population oflymphocytes bearing receptors for Fc and C3, but lacking mlg or SRBC receptors. It was shown, in a previous study, that the majority of bovine lymphocytes (87°Jo) having Fc receptors also possessed mlg, and the percentages of cells having mlg and Fc receptors closely paralleled each other in studies of 24 clinically normal cattle [7]. Further when a combined E- and EA-rosette assay for simultaneously detecting presumptive T and B cells was conducted, sub-populations of cells were found with both receptors (1.25~o) and no receptors (21.2~,). In the present study only 0.6~o+0.5 of lymphocytes with adherent SRBC also possessed mlg. An E-rosette forming cell was defined as a lymphocyte with as least two adherent SRBC, whereas, others have defined it as a cell with more adherent SRBC. While this tends to increase the percentage of E-rosettes counted, the data make it apparent that the majority of these cells have five or more adherent nSRBC. With most species studied thus far, optimal rosette formation is very much dependent upon procedural techniques. Modifications of the E-rosette assay reported herein may be found which will further improve the accuracy of the test. Ongoing investigations to enhance the precision of the T- and B-cell enumerations are directed at development of a modification, which would allow for specific identification of monocytes in PBL preparations. With the development of reliable techniques for their enumeration, major advances should be forthcoming in understanding the role T and B lymphocytes play in immune expression in both normal and diseased cattle.
REFERENCES 1. Higgins,D. A. and Stack, M. J., Bovinelymphocytes:recognitionof cells formingspontaneous (E) rosettes, Clin. exp. Immunol. 27, 348 354 (1977). 2. Fruchtmann, R., Uhlenbruck, G., Schmid, D. V. and Cwik, S., Zur charakterisierung yon T- und Blymphozyten beim rind, Zentbl. Vet Med. B. 24, 486-496 (1977). 3. Grewal, A. S., Rous, B. T. and Babiuk, L. A., Erythrocyterosettes a marker for bovineT cells, Can. J. comp. Med. 40, 298-305 (1976). 4. Binns, R. M., Sheep erythrocyte rosettes in pigs, sheep, cattle and goats demonstrated in the presence of dextran, J. lmmunol. Method~ 21, 197-210 (1978). 5. Grewal, A. S. and Babiuk, L. A., BovineT-lymphocytes an improved technique of E-rosette formation, J. lmmunol. Methods 24, 355-361 (1978). 6. Paul, P. S., Senogles, D. R., Muscoplat, C. C. and Johnson, D. W., Enumeration of T cells, B cells and monocytes in peripheral blood of normal and lymphocyticcattle, Clin. exp. Immunol. 35, 306-316 (1979). 7. Reeves,J. H. and Renshaw, H. W., Surfacemembrane markers on bovine peripheral blood lymphocytes,Am. J. vet. Res. 39, 917-923 (1977). 8. Wardley, R., An improved E-rosette technique for cattle, Br. vet. J. 133, 432 434 (1977). 9. Wilson, A. B. and Coombs, R. R. A., Rosette-formation between guinea pig lymphoid cells and rabbit erythrocytes--a possible T-cell marker, Int. Archs Allergy appl. Immunol. 44, 544-552 (1973).
86
tIARLAND W. RI:NSItAW
10. Herbert, W. J., Passive haemagglutination. In Handbook O/' Evperimental Immunology, Weir, D. W. (ed.), Blackwell Scientific, Oxford pp. 720 744 (1967). 11. B6yum, A., Isolation of mononuclear cells and granulocytes from human blood, Stand. J. olin. Lab. Inves't. Suppl. 97, 77 89 (1968). 12. Hummeler, K., Harris, T. N., Tomassini, N., Hechtel, M. and Farber, M. B., Electron microscopic observations on antibody-producing cells in lymph and blood, J. exp. Med. 12.4, 255 262 (1966). 13. Aiuti, F., Cerottini, J.-C., Coombs, R. R. A., Cooper, M., Dickler, H. B., Froland, S. S., Fudenberg, H. H. Greaves, M. F., Gray, H. M., Kunkel, H. G., Natvig, J. B., Preud'Homme, J.-L., Rabellino, Ritts, R. E., Rowe, D. S., Seligmann, M., Siegal, F. P., Stjernsward, L, Terry, W. D. and Wybran, J., Identification, enumeration, and isolation of B and T lymphocytes from human peripheral blood, Stand. J. Immunol. 3, 521-532 (1974). 14. Weiner, M. S., Bianco, C. and Nussenzweig, V., Enhanced binding of neuraminidase-treated sheep erythrocytes to human T lymphocytes, Blood 42, 939-946 (1973). 15. Bentwich, Z., Douglas, S. D., Skutelsky, E. and Kunkel, H. G., Sheep red cell binding to human lymphocytes treated with neuraminidase: enhancement of T cell binding and identification of a subpopulation of B cells, J. exp. Med. 137, 1532 1537 (1973). 16. Galili, U. and Schlesinger, M., The formation of stable E rosettes after neuraminidase treatment of either human peripheral blood lymphocytes or of sheep red blood cells, J. lmmunol. 112, 1628 1634 (1974). 17. Han, T. and Minowada, J., Enhanced E- and EAC-rosette formation by neuraminidase, J. Immunol. Methods 12, 253 260 (1976). 18. Hallberg, T., Human lymphocyte subpopulations, Acta path. microbiol, stand. B. Suppl. 250, 1 54 (1975). 19. Johansen, K. S., Johansen, T. S. and Talmage, D. W., T cell rosette formation in primates, pigs, and guinea pigs. The influence of immunosuppressive agents, J. Allergy olin. lmmunol. 54, 86 93 (1974). 20. Terrell, T. G., Holmberg, C. A. and Osburn, B. I., Immunologic surface markers on nonhuman primate lymphocytes, Am. J. vet. Re.s. 38, 503 507 (1977). 21. Mac Key, L. J., Distribution o f t and B cells in thymus, blood and lymph nodes of the cat, Res. vet. Sci. 22,225 228 (1977). 22. Bowles, C. A., White, G. S. and Lucas, D., Rosette formation by canine peripheral blood lymphocytes. J. hnmunol. 114, 399 402 (1975). 23. Escajadillo, C. and Binns, R. M., Rosette formation with sheep erythrocytes--A possible T-cell marker in the pig, Int. Archs Allergy appl. Immunol. 48, 261 275 (1975). 24. Shimizu, M., Pan, I. C. and Hess, W. R., T and B lymphocytes in porcine blood, Am. J. vet. Res. 37, 309 317 (1976). 25. Tart, M. J., Olsen, R. G , Krakowka, G. S., Cockerell, G. L. and Gabel, A. A., Erythrocyte rosette formation of equine peripheral blood lymphocytes, Am. J. vet. Res. 38, 1775 1779 (1977). 26. 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H., Rosette formation by guinea pig thymocytes and thymus derived lymphocytes with rabbit red blood cells, J. lmmunol. 111, 1834 1837 (1973). 31. Kenyon, S. J. and Piper, C. E., Cellular basis of persistent lymphocytosis in cattle infected with bovine leukemia virus, Inject. lmmun. 16, 891 897 (1977). 32. Muscoplat, C. C., Johnson, D. W., Pomeroy, K. A., Olson, J. M., Larson, V. L., Stevens, J. B. and Sorensen, D. K., Lymphocyte surface immunoglobulin : frequency in normal and lymphocytic cattle, Am. J. vet. Res. 35, 593 595 (1974). 33. Muscoplat, C. C., Johnson, D. W., Pomeroy, K. A., Olson, J. M., Larson, V. L. Stevens, J. B. and Sorensen, D. K. Lymphocyte subpopulations and immunodeficiency in calves with acute lymphocytic leukemia, Am. J. vet. Res. 35, 1571 1573 (1974). 34. Muscoplat, C. C., Johnson, D. W. and Teuscber, E., Surface immunoglobulin of circulating lymphocytes in chronic bovine diarrhea: abnormalities in cell populations and cell function, Am. J. vet. 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