Direct fluorescent labeling of cells with fluorescein or rhodamine isothiocyanate. II. Potential application to studies of lymphocyte migration and maturation

Journal of Immunological Methods, 37 (1980) 109--121 © Elsevier/North-Holland Biomedical Press

109

DIRECT F L U O R E S C E N T LABELING OF CELLS WITH FLUORESCEIN OR RHODAMINE ISOTHIOCYANATE. II. POTENTIAL APPLICATION TO STUDIES OF LYMPHOCYTE MIGRATION A N D MATURATION i

EUGENE C. BUTCHER 2, ROLAND G. SCOLLAY 3 and IRVING L. WEISSMAN 4

Laboratory of Experimental Oncology, Department of Pathology, Stanford University Medical Center, Stanford, CA 94305, U.S.A. (Received 12 February 1980, accepted 1 May 1980)

The effect of direct cell labeling with fluorescein or tetramethyl rhodamine isothiocyanate on lymphocyte migration is examined. In vitro conditions of labeling are defined which (1) do not significantly affect immediate or long term viability of lymphocytes (up to 2 weeks after transfer in vivo), (2) do not alter normal lymphocyte migration, (3) do not affect expression or detectability of surface antigens, and (4) permit direct visualization and counter-staining with fluorescent antibody reagents for days after intravenous injection. The potential application of this method to studies of lymphocyte migration and maturation is discussed.

INTRODUCTION

Studies of the phenotype, functional capacity, and migratory characteristics of lymphocytes from various organs suggest that different subsets of lymphocytes may have separate, clearly defined migration pathways (Gutman and Weissman, 1973; Sprent, 1973; Guy-Grand et al., 1974; Weissman et al., 1974; Cantor and Weissman, 1976; Butcher and Weissman, 1980a). It has also been proposed that lymphocytes follow defined maturation sequences that can be monitored by virtue of sequential expression of surface antigens (Cantor and Weissman, 1976). Current methods of lymphocyte labeling using radioisotopes do not lend themselves easily to the investigation of in vivo migration and maturation of certain lymphocyte s u b p o p u l a t i o n s particularly antigenically defined subpopulations of small lymphocytes. Most radioisotopes fail to label all cell types equally. Individual radio-labeled cells cannot be identified while alive. Furthermore, although methods are I Supported by U.S.P.H.S. Grant AI 09072. 2 To whom reprint requests should be addressed. Recipient of NIH Postdoctoral Training Grant, GM 002236-04 in Experimental Pathology. 3 Special Fellow of the Leukemia Society of America. Present address: Walter and Eliza Hall Institute, Melbourne, Australia. 4 Faculty Research Awardee of the American Cancer Society.

110 available for defining the antigenic p h e n o t y p e of labeled lymphocytes (e.g. antibody mediated c y t o t o x i c i t y or fluorescence activated cell sorting followed by autoradiography), these methods are remarkably tedious. In the accompanying paper we have described a method of covalently labeling viable cells with the isothiocyanate derivatives of fluorescein (FITC) or rhodamine {RITC) (Butcher and Weissman, 1980b). Labeled cells can be easily identified by fluorescence microscopy or fluorescence activated cell sorter analysis, and their antigenic p h e n o t y p e can be determined by counter-staining with fluorescent antibody reagents of a different color. In the present paper, we explore the possibility of applying these techniques to the study of in vivo l y m p h o c y t e migration and maturation. MATERIALS AND METHODS Animals (Balb/c X C57BL/6J) F1 hybrids, BALB/c.H-2 b, C57BL/10J and C57BL/ Ka mice were used. C57BL/Ka-Thy 1.1 mice were bred and maintained in our colony. The latter strain was kindly supplied b y Dr. Miriam Leiberman. Cell suspending procedure and medium As previously described (Butcher and Weissman, 1980b), cell suspending medium (CSM) was 5% fetal calf serum in an equal mixture of Medium 199 (Grand Island Biological Company) and Dulbecco's phosphate buffered saline, pH 7.4. Suspensions were prepared by pressing minced organs through 200 gauge stainless steel screen. Cell labeling with FITC or R I T C Preparation of stock solutions of FITC and RITC and standard labeling procedures have been previously described (Butcher and Weissman, 1980b). Details of labeling conditions in each experiment are provided in figure and table legends. Fluorescence activated cell sorter (FACS) analysis A Becton Dickinson FACS-III was used to analyze and quantitate cellular fluorescence, as previously described (Butcher and Weissman, 1980b), except that in several experiments units of fluorescence were determined by comparison with previously analyzed FITC-labeled lymphocytes (from the homing experiment in Fig. 2} rather than with fixed chicken red blood cells. Cell labeling with chromium-51 and i n d i u m - I l l L y m p h o c y t e s were labeled at 5 X 107 cells/ml by modifications of standard techniques (Rannie et al., 1977; Ford, 1978; Frost et al., 1978), either

III

with 50--100 pCi/ml Na2SlCrO4 (New England Nuclear, Boston MA) for 70 min at 37°C in CSM, or with about 30 pCi/ml i n d i u m - I l l oxine (Diagnostic Isotopes, Bloomfield, NJ) for 15 min at room temperature in a 1 : 1 mixture of normal saline (used to dilute the radioisotope) and Medium 199 with 5% fetal calf serum (containing the cells).

In vivo homing studies using S~Cr-labeled sample cells Two methods of comparing the short-term homing ability of normal and FITC- or RITC-labeled lymphocytes were employed. In one experiment, SlCr-labeled lymphocytes labeled with various levels of FITC were injected i.v. into recipients and the percentages of injected counts localizing in various organs were determined. Four recipients were used per sample. In another experiment, each SlCr-labeled sample was mixed with a 11~Indlabeled population of lymphocytes prior to i.v. injection. The ~11Ind-labeled cells served as an internal standard, reducing or eliminating errors due to injection volume or differences between individual animals in organ size and blood flow to various organs. The ratio of cpm SlCr (associated with sample cells, unlabeled or labeled with various levels of FITC or RITC) to cpm ~'~Ind (associated with standard cells) was determined in the injected cell mixtures (R~n~) and in various organs in the recipients killed 2 h after injection. The mean ratio of S~Cr to l ~ I n d in each organ (Rorgan) was determined for the two recipients of each SlCr-labeled cell population. A specific localization ratio (SLR), Rorgan/Rinj, was calculated for each sample (SLRA,B,c...), relating the localization of these SlCr-labeled populations to that of the l~Ind-labeled internal standard population. Finally, the localization of each of the S~Cr-tagged FITC- or RITC-labeled sample populations was expressed as the percentage localization relative to that of a (S~Crtagged) non-fluorescent control population: % localization of sample A relative to unlabeled control -

SLRA

SLRcontrol

× 100.

The details of the cell labeling and injection protocols are presented in the figure and table legends.

Fluorescence microscopy o f cell suspensions and frozen sections A Zeiss microscope equipped w,ith ultraviolet epi-illumination and exciter/ barrier filter combinations for fluorescein and rhodamine was used for microscopy. Cells were suspended in CSM or fixed in slightly hypertonic PBS (1.25× concentrated) containing 1% formaldehyde and examined under coverslips. For histologic localization of fluorescent cells, 5--10 pm frozen sections of lymphoid organs were examined either air-dried and uncovered, or m o u n t e d under coverslips with a 5 : 1 mixture of glycerol and PBS after 10 min soaking in PBS to remove diffusible label.

112

Suspension staining for identification of B cells or Thy 1.2 bearing cells Cell suspensions were stained using standard techniques with fluoresceinated or rhodaminated rabbit anti-mouse immunoglobulin, or with a first stage monoclonal rat antibody against T h y 1.2 (H12, kindly supplied b y J. Ledbetter) followed by a highly specific fluoresceinated rabbit anti-rat immunoglobulin second stage. RESULTS

(I) Migratory patterns of labeled lymphocytes After i.v. injection, heavily labeled fluoresceinated or rhodaminated l y m p h o c y t e s were easily visible in frozen sections or cell suspensions of lymphoid organs, and they homed to these organs in a qualitatively normal fashion. Labeled cells rapidly localized in the post-capillary high endothelial venules of lymph nodes and Peyer's patches, and in the marginal sinus of the spleen, and they subsequently migrated into B and T cell domains (Fig. 1) (see Weissman et al., 1974). They migrated in large numbers into the thoracic duct (i.e. 20% of thoracic duct l y m p h o c y t e s were fluorescent 12 h after i.v. injection of a b o u t 6 × 107 FITC-labeled spleen cells), suggesting that recirculation was normal as well.

(II) Quantitative comparison of migration of labeled and unlabeled cells To determine if fluorescent labeling quantitatively altered l y m p h o c y t e homing, we studied the in vivo localization of SlCr-tagged mesenteric node l y m p h o c y t e s labeled with increasing concentrations of FITC and RITC. As shown in Fig. 2, above a certain level of fluorescence, both RITC and FITC progressively inhibited the normal localization of l y m p h o c y t e s in lymph nodes, Peyer's patches, and spleen 3 h after i.v. injection. Correspondingly, localization in the liver, a site known to collect damaged cells, was increased. (These effects were probably n o t simply due to cell death, since we have previously failed to observe a significant effect of FITC or RITC labeling on short term cell viability even at the highest levels used in this experiment.) This experiment permits confident definition of the conditions of labeling and the m a x i m u m median cellular fluorescence permissible in quantitative studies of short term l y m p h o c y t e migration, i.e. a b o u t 300 fluorescence units/cell fluorescein, or 45 units/cell rhodamine. A further experiment was performed to determine if localization at lower levels of fluo~resceination was still unaffected 24 h after injection. At the median levels of fluorescence used (380 and 102 units fluorescein, similar to those of the lowest two labeled groups in the previous experiment, Fig. 2), l y m p h o c y t e localization was not detectably altered (Table 1).

113

Fig. 1. Homing of labeled lymphocytes visualized by fluorescence microscopy in frozen sections of lymph nodes. A: Localization in post-capillary high endothelial venules within minutes after i.v. injection. B: Subsequent migration into the lymph node parenchyma; 30 min after injection. Approx. X 200.

(III) Long term viability and visibility o f FITC- and RITC-labeled lymphocytes after i.v. injection Having d e f i n e d levels o f cellular f l u o r e s c e n c e t h a t did n o t significantly alter s h o r t t e r m l y m p h o c y t e m i g r a t i o n , we t h e n asked if these levels were

114 '-5

~

Io

""

/

I I I 26 44 57 MEDIAN FLUORESCENCE

I

I J'-'f~,r

lO7 I Io /

/

I t I 114 310 715 MEDIAN FLUORESCENCE

E

s0 -

0

0 .-.f.L

\

I 1.5

I 3

I 1327

R

\,

l 6

I-g 12

~, O

[RITC] pg/ml

I 30

I 60

I

"ID

120

300

[FITC] pg/ml

Fig. 2. T h e e f f e c t o f labeling w i t h various levels of R I T C a n d F I T C o n t h e s h o r t t e r m in vivo h o m i n g o f l y m p h o c y t e s , s i Cr-tagged m o u s e l y m p h n o d e cells were labeled w i t h 0 - - 3 0 0 p g / m l F I T C (15 m i n at r o o m t e m p e r a t u r e at 5 × 1 0 ? / m l in CSM a d j u s t e d t o pH 7.0 w i t h HC1) or 0 - - 1 5 # g / m l R I T C (15 m i n , r o o m t e m p e r a t u r e at 5 × 1 0 7 / m l in CSM, p H 7.4), w a s h e d a n d i n j e c t e d i n t r a v e n o u s l y i n t o s y n g e n e i c age- a n d s e x - m a t c h e d r e c i p i e n t s along w i t h an l l I i n d _ t a g g e d i n t e r n a l s t a n d a r d p o p u l a t i o n of l y m p h o c y t e s . L o c a l i z a t i o n in spleen, axillary a n d b r a c h i a l l y m p h n o d e s , P e y e r ' s p a t c h e s a n d liver was d e t e r m i n e d 2 h later. T h e l o c a l i z a t i o n of (SICr-tagged) FITC- or R I T C - l a b e l e d l y m p h o c y t e s in e a c h o r g a n is e x p r e s s e d as a p e r c e n t a g e o f t h e l o c a l i z a t i o n o f (SlCr.tagged) u n l a b e l e d cells. E a c h p o i n t is t h e m e a n of t w o r e c i p i e n t s .

TABLE 1 E f f e c t o f F I T C labeling o n in vivo l y m p h o c y t e m i g r a t i o n p a t t e r n s . S iCr-tagged l y m p h n o d e cells were labeled w i t h 0, 30 or 60 p g / m l F I T C ( 3 7 ° C 20 m i n at 5 × 1 0 7 / m l in CSM, p H 7.4) a n d i n j e c t e d i.v. i n t o age- a n d s e x - m a t c h e d s y n g e n e i c recipients. R e c i p i e n t s were killed 3 or 23 h l a t e r a n d t h e r a d i o a c t i v i t y in spleen, p e r i p h e r a l l y m p h n o d e s (PLN), small i n t e s t i n e (SI, i n c l u d i n g P e y e r ' s p a t c h e s ) , liver a n d l u n g was d e t e r m i n e d . Hours after injection

Organ

F I T C labeling c o n c e n t r a t i o n ( u n i t s f l u o r e s c e n c e )

o(o)

23

30/Jg/ml (102)

60/zg/ml (380)

Spleen PLN SI Liver Lung

19.0 0.8 1.5 26.1 4.6

+ 1.0 a -+ 0.1 -+ 0.1 -+ 1.0 -+ 0.3

19.6 0.9 1.5 24.1 5.1

-+ 0.5 -+ 0.1 -+ 0.1 -+ 0.5 -+ 0.3

18.7 0.9 1.3 23.0 3.9

-+ 0.7 + 0.0 -+ 0.2 + 0.2 -+ 0.1

Spleen PLN SI Liver Lung

14.3 1.1 1.3 22.0 0.8

-+ 0.7 -+ 0.1 + 0.1 -+.0.7 -+ 0.1

16.7 1.5 1.5 22.8 1.1

-+ 0.6 -+ 0.1 -+ 0.0 -+ 0.3 -+ 0.1

15.2 1.3 1.5 22.8 1.0

-+ 0.8 -+ 0.1 -+ 0.0 -+ 0.8 -+ 0.1

a M e a n a n d s t a n d a r d e r r o r o f t h e p e r c e n t a g e s o f i n j e c t e d c o u n t s f o u n d in t h e organs o f 4 i n d i v i d u a l recipients.

115

adequate for experimental use -- i.e. could intravenously injected labeled cells be visualized in frozen sections or cell suspensions of lymphoid organs and, if so, for how long? Five X 107 viable spleen and mesenteric node lymphocytes labeled with FITC (median fluorescence 234) or RITC (median fluorescence 32) were injected intravenously into syngeneic recipients. Recipients were killed 19 h to 11 days later and frozen sections or cell suspensions of spleen and lymph nodes were examined for the presence of fluorescent cells. Histologic localization: visibility in frozen sections. In frozen sections, the RITC-labeled cells were difficult to visualize even at the earliest time point (19 h after injection). On the other hand, the FITC-labeled cells were easily identified at this time distributed randomly in lymph nodes and in the white pulp of the spleen. The brightness of the FITC-labeled cells declined progressively with time, but numerous cells could still be observed 11 days after injection. FITC-labeled cells localizing in B cell areas appeared on average significantly brighter than those in T cell domains, as would be predicted from our previous report that B cells are more heavily labeled with FITC than T cells (Butcher and Weissman, 1980b). No such regional difference was observed with RITC-labeled cells. Visibility in cell suspensions. Both RITC- and FITC-labeled cells were easily visualized in cell suspensions even after 11 days in vivo. Two to three days after injection, their fluorescence had faded to such a degree that a 40X objective was required to permit confident identification. At later time points (5--11 days) and in contrast to the frozen section results, RITC-labeled cells were easily enumerable than FITC-labeled cells, probably because the former are more uniformly labeled. The frequency and brightness of FITC-labeled cells, and the frequency of RITC-labeled cells among mesenteric node lymphocytes (determined b y direct counting under fluorescence microscopy, and/or b y FACS analysis) are presented in Table 2 . (FACS analysis of the RITC-labeled cells was n o t possible because of the inefficiency of our laser and optical system in the required wave lengths.) The data demonstrate that (1) the behavior of RITC- and FITC-labeled cells was similar; (2) the frequency of identifiable RITC- and FITC.labeled cells declined slowly, so that only a b o u t 50% as many cells were found at 11 days as at 19 h; and (3) as previously demonstrated in vitro at 37°C (Butcher and Weissman, 1980b), the median fluorescence of labeled cells declined rapidly in the first day (to a b o u t 4% of its initial value), and more slowly thereafter. To determine if the decline in cell number with time was due to fading, to cell damage secondary to the label, or to the natural lifespan of injected cells we repeated the long term homing experiment using cells carrying an antigenic surface marker, Thy 1.2, thus allowing direct comparison of FITCor RITC-labeled cells with unlabeled cells. The frequency of Thy 1.2 positive (donor) T cells in recipient (Thy 1.1) mesenteric nodes was determined by FACS analysis of cell suspensions stained with specific monoclonal antibody, and was quantitatively confirmed for several recipients by direct fluores-

116 TABLE 2 L o n g - t e r m visibility o f l a b e l e d l y m p h o c y t e s in vivo. T h e results are e x p r e s s e d as p e r c e n t ages o f visibly f l u o r e s c e n t l y m p h o c y t e s in r e c i p i e n t m e s e n t e r i c n o d e a t various t i m e s a f t e r i n j e c t i o n . I n d i v i d u a l r e c i p i e n t s were killed at various t i m e s a f t e r i n j e c t i o n of 5 × 107 syngeneic, viable, FITC- or R I T C - l a b e l e d spleen a n d m e s e n t e r i c n o d e l y m p h o c y t e s . T h e % of f l u o r e s c e n t cells in t h e r e c i p i e n t m e s e n t e r i c l y m p h n o d e was d e t e r m i n e d m i c r o s c o p i c a l l y or b y F A C S analysis. Days a f t e r injection

0.8 2 3 5 11

% F l u o r e s c e n t cells RITC a

FITC b

Microscope

Microscope

FACS

Median fluorescence (% of initial value)

4.7±0.6 4.8±0.6 3.2±0.4 ND 2.1±0.3

4.7±0.5 3.9±0.1 ND 2.9±0.4 2.4±0.2

5.1 4.3 3.7 -2.4

4.0 3.4 2.8

c

0.9

a 2 p g / m l R I T C , 2 ×107 cells/ml, 15 m i n at r o o m t e m p e r a t u r e in CSM. M e d i a n fluorescence 32. b 35 p g / m l F I T C , 5 × 107 cells/ml, 17 m i n a t r o o m t e m p e r a t u r e in CSM, pH 7.0. M e d i a n f l u o r e s c e n c e 234. c % f l u o r e s c e n t cells a n d s t a n d a r d error. TABLE 3 L o n g - t e r m survival o f heavily labeled l y m p h o c y t e s in vivo: c o m p a r i s o n w i t h u n l a b e l e d l y m p h o c y t e s . Five × 107 u n l a b e l e d , F I T C - l a b e l e d , or R I T C - l a b e l e d C 5 7 B L / 1 0 ( T h y 1.2) spleen a n d m e s e n t e r i c l y m p h n o d e ( M L N ) cells were i n j e c t e d i.v. i n t o C 5 7 B L / K a - T h y 1.1 recipients. T w o w e e k s l a t e r t h e p e r c e n t a g e o f d o n o r cells, d e t e c t e d w i t h m o n o c l o n a l antiT h y 1.2 a n t i b o d y , was d e t e r m i n e d m i c r o s c o p i c a l l y or w i t h t h e F A C S in cell s u s p e n s i o n s o f r e c i p i e n t m e s e n t e r i c nodes. Label

Recipient

% D o n o r cells in M L N

Mean and standard error

None

1 2 3

2.4 2.6 2.6

2.5 -+ 0.1

4 5 6

1.8 2.7 3.3

2.6 -+ 0.4

7 8 9

1.0 3.4 1.6

2.0 + 0.7

RITC a

FITC b

a 3.4 p g / m l R I T C , 3 × 107 cells/ml, 15 m i n at r o o m t e m p e r a t u r e in CSM. M e d i a n fluorescence 51. b 46 p g / m l F I T C , 3 × 107 cells/ml, 17 m i n at r o o m t e m p e r a t u r e in CSM, pH 7.0. M e d i a n f l u o r e s c e n c e 368.

117

cence microscopy. The residual fluorescence of the RITC- and FITC-labeled cells was insignificant compared to that due to the anti-Thy 1.2 staining. The experiment failed to demonstrate a selective loss of labeled cells compared with the unlabeled controls (Table 3). Since our anti-Thy 1.2 indicator was fluorescein (second stage fluoresceinated rabbit anti-rat immunoglobulin), we were able to assess directly the presence of Thy 1.2 and RITC label on individual cells. Sixty-four % (100 out of 156) of rhodamine positive cells were Thy 1.2 positive; this percentage reflects that of T cells in the mesenteric node {60--70%) (as would be predicted since the injected pooled spleen and mesenteric node l y m p h o c y t e suspension probably contained roughly equal numbers of B and T cells). Only 5% of Thy 1.2 positive cells could not be confidently assessed as rhodamine positive (5 of 107). These data indicate that the RITC-labeled cells are truly of donor origin, that RITC-labeled B cells are probably as stable as T cells, and that fading or selective cell death due to label is probably not a major factor in the decline of detectable labeled cells with time.

TABLE 4 In vivo h o m i n g of surface Ig positive a n d negative l y m p h o c y t e s labeled w i t h various levels of F I T C a n d R I T C . B A L B / c . H-2 b spleen cells ( c o n t a i n i n g 50.4% surface Ig bearing cells) f r o m 10-week-old male d o n o r s were labeled in CSM at 6 x 107 cells/ml for 15 m i n w i t h F I T C (at 3 7 ° C ) o r R I T C ( a t r o o m t e m p . ) a n d washed. Five x 107 labeled cells were i n j e c t e d i.v. i n t o s y n g e n e i c 8 - - 1 0 - w e e k - o l d f e m a l e r e c i p i e n t s w h i c h were killed 2 h later. S u s p e n s i o n s o f r e c i p i e n t l y m p h n o d e (axillary, brachial a n d superficial cervical) a n d spleen cells were s t a i n e d w i t h e i t h e r f l u o r e s c e i n a t e d or r h o d a m i n a t e d r a b b i t a n t i - m o u s e i m m u n o g l o b u l i n a n t i s e r u m as a p p r o p r i a t e . FITC- or R I T C - l a b e l e d ( d o n o r ) cells were i d e n t i f i e d using filters for t h e a p p r o p r i a t e f l u o r o c h r o m e , a n d s c o r e d as Ig ÷ or Ig- b y s w i t c h i n g filter c o m b i n a t i o n s t o visualize t h e f l u o r e s c e n t a n t i - i m m u n o g l o b u l i n stain o f t h e o t h e r color. Label

FITC

RITC

Concentration (pg/ml)

42 32 21 3.4 1.7 0.8 0.4

U n i t s cellular fluorescence (median)

290 197 94 18 11 5.6 3.5

% I n j e c t e d cells b e a r i n g surface Ig in r e c i p i e n t s ' lymph node

spleen

23.8 -+ 2.4 a 18.2 -+ 2.2 23.2 -+ 2.9

50.0 -+ 4.4 ND b 46.3 -+ 4.6

18.5 22.2 20.4 22.1

54.0 + 4.7 ND ND 48.7 + 4.5

a P e r c e n t a g e Ig positive d o n o r cells, a n d s t a n d a r d error. b Not determined.

+ 2.0 + 2.8 + 2.4 -+ 2.6

118

(IV) Effect o f label on short term migration o f B and T lymphocytes To determine if fluorescent label selectively alters the migration of l y m p h o c y t e subpopulations, we compared the relative localization of surface Ig positive and Ig negative spleen cells after labeling with various levels of FITC or RITC. Levels of labeling were chosen with no detectable effect on the homing of whole l y m p h o c y t e populations, as defined by Fig. 1 and Table 1. Two hours after injection into syngeneic hosts, suspensions of the recipients' mesenteric nodes and spleen were made and the percentage of surface Ig bearing donor cells was determined by counter-staining with rhodamine or fluorescein conjugated rabbit anti-mouse immunoglobulin. The results, presented in Table 4, demonstrate that the relative localization of Ig positive and Ig negative spleen cells in these organs was unchanged by labeling with a wide range of concentrations of FITC or RITC, indicating that these levels of labeling do not selectively alter the migration of these l y m p h o c y t e subclasses. It is apparent that both Ig bearing and Ig negative cells localize well in the spleen (roughly 50% of donor cells in the spleen had surface Ig, about the same proportion as in the injected population), whereas Ig negative cells home selectively to the lymph node (only 20% of donor cells in the node had surface Ig). DISCUSSION The experiments we have presented define levels of labeling with FITC and RITC that do not alter short term l y m p h o c y t e homing, and demonstrate that l y m p h o c y t e s so labeled migrate normally, recirculate and remain viable and visibly fluorescent for days in vivo. Table 5 presents a summary of some of the similarities and differences between FITC and RITC as cell labels, and between the characteristics of FITC- and RITC-labeled cells. Although both cell labels are suitable for short term migration experiments (thus making it possible to trace two subpopulations in the same recipient), their differences make each one preferable for certain applications. For instance, RITC might be preferred for quantitative studies of the short and long term migration of l y m p h o c y t e s to be counter-stained with specific antisera in cell suspensions of recipient organs, since RITC-labeled cells are apparently labeled w i t h o u t regard to subtype and are easily visible in cell suspensions even at very low levels of labeling. FITC may be better for experiments requiring histologic localization of cells, since fluoresceinlabeled cells are more easily visible in frozen sections (at least with the microscopic facilities we have employed). FITC would also be preferred in experiments requiring mixing of labeled and unlabeled cells in vitro prior to injection, since RITC-labeled cells could transfer fluorescence to the unlabeled cells (Butcher and Weissman, 1980b). (It should be stressed, however, that although RITC-labeled cells transfer label to unlabeled cells in vitro, this is not a problem when they are diluted by injection in vivo;

119 TABLE 5 Comparison of FITC and RITC labeling for studies of in vivo lymphocyte migration. FITC

RITC

Conditions of labeling

~<40 pg/ml 37°C 15 min in CSM at 5 × 107 cells/ml

< 4 pg/ml 25°C 15 min in CSM at 5 × 107 cells/ml

Uniformity of label

B cells brighter than T cells

Good (fluorescence proportional to cell size)

Effect of label on: Cell viability GVH response In vivo migration

None detected None detected None detected

None detected None detected None detected

Visibility after injection in vivo In frozen sections In cell suspensions

Fair Good

Poor Excellent (visible even after 14 days when heavily labeled)

Transfer of label to bystander cells In vitro In vivo (after i.v. injection)

None detected None detected

Significant None detected

None detected (Ig, Thy 1, ThB, Lyt-1, Lyt-2, H-2D d, H-2K k, IA k )

None detected (Ig, Thy 1, others not tested)

Effect on surface antigens

counter-staining with anti-Thy 1.2, for instance, confirmed that the expected proportion of RITC-labeled cells expressed the donor Thy 1 when labeled cells were injected into Thy 1.1 recipients.) Using our reagents, the conditions of labeling presented in Table 5 yield the m a x i m u m r e c o m m e n d e d levels of fluorescence, as defined by the experiments in this paper. Since batches of FITC and RITC differ, these protocols should be used only as an approximate guide. New reagents should be standardized by determination of the minimal conditions that inhibit migration. Several important points are illustrated by the experiment comparing the localization of Ig ÷ and Ig- lymphocytes 2 h after i.v. injection. Firstly, cell labeling is effective over a wide range of fluorochrome concentrations, and at levels much below those affecting l y m p h o c y t e migration (i.e., satisfactory cellular fluorescence was obtained at 1/3 the concentration of FITC, and 1/10 that of RITC, that inhibited migration in Fig. 2). Secondly, neither FITC nor RITC selectively alters the homing of the major l y m p h o c y t e subclasses, B and T cells, at the levels of labeling employed. Thirdly, neither FITC nor RITC interferes significantly with the antigenicity or detectability

120 of surface immunoglobulin. In other experiments (Scollay et al., 1978, 1980), we have f o u n d that FITC label has no effect on the detectability of any surface antigen we have examined (Ig, Thy 1, Lyt-1, Lyt-2, H-2K, IA). This is n o t surprising, since at the levels of FITC used only about 107 FITC molecules are associated with each l y m p h o c y t e , and most of these are probably internal (Butcher and Weissman, 1980b, and M. Loken, personal communication). Finally this experiment graphically demonstrates the ease with which the migratory behavior of defined l y m p h o c y t e populations can be studied with either FITC or RITC as fluorescent markers. While both surface Ig bearing (B) cells and Ig negative cells home well to the spleen, Ig negative cells localize preferentially in lymph nodes. These results directly confirm previous studies of B and T l y m p h o c y t e migration utilizing radioactive tracers (Howard et al., 1972; G u t m a n and Weissman, 1973; Sprent, 1973). Although we have limited this paper to discussion of in vitro cell labeling, FITC and RITC can also be used to label lymphocytes and other cells topically in vivo. Thus, we have employed intrathymic microinjection of FITC to label t h y m o c y t e s in situ, and have been able to identify and quantitate the rate of appearance of thymic migrants in the periphery. Their antigenic p h e n o t y p e was characterized by counter-staining with appropriate antisera. Similar experiments have been performed using intrasplenic administration of FITC. These methods are described in detail elsewhere (Scollay et al., 1978, 1980). In summary, lymphocytes labeled by covalent reaction with FITC or RITC under benign conditions migrate normally in vivo, and can be identified and antigenically characterized for days after i.v. injection. Application of this technique to the study of the migration of antigenically defined l y m p h o c y t e populations will increase our understanding of the mechanism and functional importance of l y m p h o c y t e recirculation, and should allow direct examination of l y m p h o c y t e maturation sequences in vivo. ACKNOWLEDGEMENTS We t h a n k S. Stevens, S. Jacobs, L. Jerabek, M. Beers, D. Chestnut and C. B u t n u t for willing assistance, and R. Coffman for his antisera. REFERENCES Butcher, E.C. and I.L. Weissman, 1980a, in: Blood Cells and Vessel Walls. Functional Interactions, CIBA Foundation Symposium 71 (Excerpta Medica, Amsterdam) p. 256. Butcher, E.C. and I.L. Weissman, 1980b, J. Immunol Methods 37, 97. Cantor, H. and I.L. Weissman, 1976, Prog. Allergy 20, 1. Ford, W.L., 1978, in: Handbook of Experimental Immunology, 3rd edit., D.M. Weir (Davis, Philadelphia) Ch. 23. Frost, P., J. Smith and H. Frost, 1978, Proc. Soc. Exp. Biol. Med. 157, 61.

121 Gutman, G.A. and I.L. Weissman, 1973, Transplantation 16,621. Guy-Grand, D., C. Griscelli and P. Vassalli, 1974, Eur. J. Immunol. 4,435. Howard, J.C., S.V. Hunt and J.L. Gowans, 1972, J. Exp. Med. 135,200. Rannie, G.H., M.L. Thakur and W.L. Ford, 1977, Clin. Exp. Immunol. 29,509. Scollay, R.G., M. Kochen, E.C. Butcher and I.L. Weissman, 1978, Nature 276, 79. Scollay, R.G., E.C. Butcher and I.L. Weissman, 1980, Eur. J. Immunol. 10, 210. Sprent, J., 1973, Cell. Immunol. 7, 10. Weissman, I.L., G.A. Gutman and S.H. Friedberg, 1974, Ser. Haemat. 7,482.