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Brief DeFinitive Report

VERY RAPID DECAY OF M A T U R E B LYMPHOCYTES IN THE SPLEEN* BY A. A. DE FREITAS AND A. COUTINHO:~

From the Department of Immunology, Umed University, Umed, Sweden; and the Department of Immunology, Medical Science Faculty, Lisboa, Portugal Classical e x p e r i m e n t s by O s m o n d a n d Nossal (1) have d e m o n s t r a t e d the c o n t i n u o u s p r o d u c t i o n o f very high n u m b e r s of B l y m p h o c y t e s in the bone m a r r o w , b u t there are few indications that a significant fraction o f these ever migrates to the p e r i p h e r y a n d p a r t i c i p a t e s in the establishment o f a v a i l a b l e repertoires. "Half-lives" of l y m p h o c y t e s have been d e t e r m i n e d b y labeling or killing d i v i d i n g cells, b u t these a p p r o a c h e s do not resolve the question o f w h e t h e r the m i t o t i c activity takes place a m o n g precursors t h a t r a p i d l y differentiate to c o m p e t e n c e a n d are e x p o r t e d to the p e r i p h e r y , or a m o n g m a t u r e , p e r i p h e r a l B lymphocytes. These two alternatives have very different consequences for the stability o f a v a i l a b l e repertoires, because the expansion of m a t u r e B cells will result in the exclusion o f novel clones p r o d u c e d in the marrow. T h i s question is f u n d a m e n t a l , because the size o f the a v a i l a b l e a n t i b o d y repertoire a p p e a r s to be p r i m a r i l y l i m i t e d b y t h e n u m b e r s o f different clones the i n d i v i d u a l contains at any given time. W e have now devised an a p p r o a c h that provides the direct m e a s u r e m e n t of the persistence o f m a t u r e B cells in the p e r i p h e r y , regardless of cell division. T h e results thus o b t a i n e d indicate average d e c a y rates of 50% p e r d a y for a large fraction of n o r m a l spleen B cells u n d e r quasi-physiologic conditions. Materials and Methods

Mice. Male C57BL/6 (B6), C57BL/10. Sc.Sn (B10.Sn), and C57BL/10.Sc.Cr. (B10.Cr) were obtained from our own colony, from Olac, Blackthorn, England, or from Bomholtgaard, Ry, Denmark, respectively. They were used between 2 and 5 mo of age. Cell Suspensions. Spleen cells were prepared as described (2), and separated by size at unit gravity in continuous gradients of bovine serum albumin (3). Lymphocyte fractions with a sedimentation velocity of 10 PFC were scored as positive, and the results were shown to conform to the first-order term of Poisson's distribution. The frequencies of reactive cells shown were * Supported by the Swedish Medical Research Council and by a grant from the Swedish Institute. :~To whom correspondence should be addressed at the Department of Immunology, Umea University, S-901 87 Umea, Sweden. 994

J. Exp. MED.© The Rockefeller University Press • 0022-1007/81/09/0994/06 $1.00 Volume 154 September 1981 994-999

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derived from semi-log plots, and indicate the numbers of cells per culture allowing 63% of the cultures to respond. Cell Transfers. The small cell fraction purified from spleen cells was washed in balanced saline solution, adjusted to 50 × 106 cells/ml and injected intravenously into recipient mice. Splenic Localization of Transferred Cells. Spleen cells were labeled with 51Cr after removal of erythrocytes and dead cells as described in detail (5). They were then transferred intravenously into normal mice and, 24 h later the recipients were killed and the amount of radioactivity recovered in the spleen was measured and expressed as a percentage of the total cellular radioactivity injected. Results a n d Discussion The mouse strain C57BL/10Sc.Cr (B 10.Cr) was previously found to carry a mutant gene at the lps locus in chromosome 4 (6) controlling the expression o f a B cell receptor for LPS from gram-negative bacteria, which can be serologically (7) and functionally (2) identified in roughly one-third of all splenic B cells. B lymphocytes of this strain fail to recognize LPS as a polyclonal mitogen. The mutation must have occurred after this substrain was separated from the related C57BL/10Sc.Sn (B10.Sn) strain that shows full reactivity to this mitogen (8). No other differences between these strains are known, and they are fully histocompatible. All attempts to induce mutual serological or cellular immunity by grafting of cells or tissues in various immunization protocols have failed thus far (A. Coutinho, unpublished results). The same applies to another related strain, C57BL/6. The strategy in these experiments was to follow the decay of LPS-responder B lymphocytes in histocompatible, untreated LPS-nonresponder recipients, using techniques that provide the direct count, by limiting dilution analysis, of all mitogenreactive cells in a population (2). Because the spleen is the major site of migration for B lymphocytes newly formed in the marrow (1), we were concerned, in the present observations, with the turnover of splenic B cells. To minimize extraneous effects caused by differential homing patterns of lymphocyte populations, we have followed LPS-responder spleen cells in the recipient's spleen. Table I shows the limits of the method, namely the frequencies of splenic B cells from B10.Cr and B10.Sn that can be activated by LPS to grow as clones of IgM-secreting PFC, as well as those measured in the absence of LPS. The latter are probably stimulated by FCS components, and their frequencies are comparable in both strains of mice and to that obtained in B 10.Cr mice in the presence of LPS (1:6,000 spleen cells), whereas in LPS-responders, 1:5 spleen cells is detected as an LPS-reactive clonal precursor. As also shown in Table I, in vitro mixtures of LPS-responder and nonresponder spleen cells, when analyzed for the frequency of LPS-reactive clones, provide the correct count; that is, the frequency expected from the independent measurements in each cell population. To avoid stem-cell contamination in the spleen cell populations to be transferred, small lymphocytes were purified (3) and used in the transfers. It is well known that viable lymphocytes, after intravenous transfer, follow specific patterns of traffic and homing (5). By 24 h, the distribution of injected lymphocytes is thought to have stabilized and we therefore took this time as the starting point in our experiments. Two independent measurements of the numbers of transferred cells homing to the spleen were taken at 24 h. One involved the conventional technique of labeling the transferred cells with radioactive chromium and determining the fraction of the injected activity recovered in the recipient spleen. Confirming previous observations

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TABLE I Frequency of LPS-reactive Cells in the Spleen of LPS-responder and LPSnonresponder Strains of Mice Experiment B10.Sn B10.Cr

LPS

Without LPS

1:5 1:6,000

1:5,500 1:6,000

1:12

ND:I:

1: 108 1:100 1:5,500

ND ND ND

I

Experiment 2 B6 B6 + B10.Gr* expected observed B10.Cr

* In mixing experiments, spleen cells from LPS-responder mice were diluted 1:9 with cells from LPS-nonresponder mice. :~Not determined. (5), the results shown in Table II demonstrate that ~20% of the injected cells are present in the spleen 24 h after transfer. Radioactivity measurements at these early times reflect viable cell counts as shown by determining the numbers of LPS-reactive (donor) B cells present in the recipient spleen at the same time. Table II also shows that 20% of the injected cells can be recovered. The removal of cells from the donor spleen and their purification and transfer certainly result in damage that influences the survival in the host. Therefore, the numbers of donor cells persisting in the host cannot be compared to the total number transferred. However, this difficulty is obviated if the numbers of donor cells found in the recipient spleen at 24 h are taken as the starting value in the experiment. Thus, injured or dead cells do not circulate and are rapidly retained in nonlymphoid organs, primarily the liver (5). T h a t is, the 24-h value represents the total numbers of cells that were successfully transferred and that will follow "normal" rates of decay in the host. This measure also corrects for the possibility that transferred cells home to organs other than spleen, as these are then excluded from the experiment. Frequency determinations of LPS-reactive cells in the recipient spleen were then performed in the days after transfer. These frequencies allowed for the calculation of the total numbers of LPS-reactive cells in the organ, and these were compared with the numbers found 24 h after transfer. Taking the fraction recovered at 24 h as the starting (100%) value, the logarithmic plot of the decay with time of LPS-reactive cells in the recipient spleen is shown in Fig. 1. These results were obtained in two independent experiments defining precisely the same curve, namely a decay rate of 50% per day. After day 5 of transfer, these methods become unsuitable, as the "background" frequencies in B10.Cr make it difficult or impossible to detect small numbers of cells. The interpretation of these results must consider first the possibility that the rapid decay rate observed represents active elimination of donor cells in nonphysiologic conditions, rather than the normal dynamics in the system. This appears unlikely, however, as no histocompatibility reactions could be detected in this strain combination, even after very intense protocols of immunization. To directly assess this

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TABLE II

Number of Lymphocytesfrom LPS-responder Mice Recoveredin the Spleen 24 h after Transfer into LPSnonresponder Mice* Frequency of LPS-reactive cells in recipient's spleen

Total number of LPSreactive cells recovered in recipient's spleen

Percent of the transferred LPS-reactive cells in recipient's spleen:~

Percent of injected 51Cr-labeledcells recovered in recipient's spleen

1:400

158,000

19,8

21.2

* All results are averages of three to five recipient mice. :~The frequency of LPS-reactive cells in the recipient's spleen was determined by limiting dilution analysis, and the number of LPS-reactive ceils recovered is expressed as a percentage of the number of LPSreactive cells injected, as assessed by limiting dilution analysis on the population of injected cells.

•~ "6 8

10.80.6-

~--

0.4-

8

0.2-

o.i._o. 0.08" (J

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0.060.04-

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"6 ..Q

E=

z Days after cell transfer

FIG. 1. Small spleen lymphocytes from LPS-responder mice were transferred intravenously into LPS-nonresponder mice. At the indicated times after transfer, recipients were killed and the frequencies of LPS-reactive B lymphocytes in spleen cells were determined. The total numbers of LPS-reactive cells remaining in the recipient spleen at the different time-points were then calculated as a fraction of the number of LPS-reactive cells found at 24 h after transfer. The figure shows the results obtained in two independent experiments. possibility, i d e n t i c a l e x p e r i m e n t s were p e r f o r m e d u s i n g e i t h e r n o r m a l r e c i p i e n t s or B 1 0 . C r mice t h a t h a d b e e n " i m m u n i z e d " 3 m o earlier w i t h 107 B6 s p l e e n cells, t h e d o n o r i n o c u l u m used in these e x p e r i m e n t s . R e s u l t s s h o w n in T a b l e III d e m o n s t r a t e t h a t b o t h t h e h o m i n g to t h e host spleen a n d t h e d e c a y in t h e o r g a n o f L P S - r e a c t i v e d o n o r B cells are n o t affected b y previous i m m u n i z a t i o n o f t h e recipients, m a k i n g it very u n l i k e l y t h a t t h e d e c a y rates o b s e r v e d are t h e result o f active e l i m i n a t i o n b y i m m u n e m e c h a n i s m s . F u r t h e r m o r e , t h e t r a n s f e r o f < 5 × 106 B ceils represents a m i n o r fraction of t h e total n u m b e r s p r o d u c e d d a i l y in the n o r m a l system a n d therefore it s h o u l d n o t b e e x p e c t e d to i n t r o d u c e gross a l t e r a t i o n s in the physiologic t u r n o v e r rates. T h e e x p e r i m e n t a l a p p r o a c h d e s c r i b e d h e r e p r o v i d e s c o n d i t i o n s t h a t a r e very little, if at all, i m m u n o g e n i c i n c o n t r a s t to p r e v i o u s l y used markers, e.g., allotypic. I n a d d i t i o n ,


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TABLE III

Recovery of LPS-responder Lymphocytesfrom the Spleens of Normal or Immunized LPS-nonresponder Hosts

Day of assay

State of recipient

Frequency of LPSreactive cells

1

Normal Immune Normal Immune

1:140 1: 125 1: 1,400 1: 1,300

4

Percent of transferred LPSreactive cells in recipients spleen at 24 h

Donor cells persisting in spleen as a fraction of 24 h value

0.29 0,3 t ---

--0.1 0.096

Normal BI0.Cr mice, or mice given one intravenous injection of l0T spleen cells from B6 mice 3 mo previously, were used as recipients of B6 spleen cells, as described in the text. 1 and 4 d after transfer, spleen ceils from groups of three mice were pooled and the frequencies of LPS-reactive cells were determined, as in Table I. Fractions of donor cells homing to the spleen at 24 h were determined as in Table II, and those of the persisting cells were determined as in Fig. 1. o u r assay requires reactivity a n d functional p e r f o r m a n c e o f the transferred cells, thus e x c l u d i n g those still alive b u t a l r e a d y u n r e a c t i v e to c o m p e t e n t stimuli t h a t c a n be detected in o t h e r protocols. Finally, the responses are s t u d i e d in vitro, a n d the functional p e r f o r m a n c e is therefore not influenced b y r e g u l a t o r y m e c h a n i s m s or o t h e r conditions specific to the host e n v i r o n m e n t . It m i g h t be a r g u e d t h a t the B cell p o p u l a t i o n defined by reactivity to L P S is not a representative s a m p l e o f the whole B cell c o m p a r t m e n t . It m a y well be t h a t d o n o r L P S - r e a c t i v e cells in our e x p e r i m e n t s d i d not in fact d e c a y in the recipient spleen, b u t simply differentiated to o t h e r stages a l o n g the p a t h w a y s o f B cell d e v e l o p m e n t with a c o n c o m i t a n t loss o f LPS-reactivity. Alternatively, L P S - r e a c t i v e cells could have m i g r a t e d out of the spleen to, for e x a m p l e , l y m p h nodes. I n these cases, however, similar decay rates w o u l d a p p l y for all stages in a given differentiative line or for the cells in the next l y m p h o i d organ, as the B cell c o m p a r t m e n t is kept r e a s o n a b l y constant in the physiologic s t e a d y state. It can be postulated, however, that L P S - r e a c t i v e l y m p h o c y t e s c o m p o s e a n indep e n d e n t subset t h a t is distinct in rate o f d e c a y from the o t h e r two-thirds o f splenic B ceils. T h i s possibility c a n n o t b e excluded, a l t h o u g h it w o u l d a p p e a r unlikely, because we have previously f o u n d that L P S - r e a c t i v e cells c o m p a r e well with the rest of the B l y m p h o c y t e c o m p a r t m e n t in the rates o f d e c a y a n d recovery, after in vivo killing o f cells in S-phase w i t h h y d r o x y u r e a (A. A. Freitas, B. R o c h a , L. F o r n i a n d A. C o u t i n h o , m a n u s c r i p t s u b m i t t e d for publication). T h e present results concern at least o n e - t h i r d o f all splenic B cells. I f they are taken as representative o f t h e m a j o r i t y o f B l y m p h o c y t e s in the spleen, it w o u l d follow t h a t ~ 2 5 × 106 B cells w o u l d leave the organ (or die) in a day. These n u m b e r s are c o m p a r a b l e to those d e t e r m i n e d b y O s m o n d a n d Nassal (1) for the rate of B cell p r o d u c t i o n in b o n e m a r r o w , a n d these observations w o u l d therefore suggest t h a t the m a j o r i t y of newly formed B cells m i g r a t e to the p e r i p h e r y a n d p a r t i c i p a t e in the astonishingly high t u r n o v e r rate described here. It should be p o i n t e d out, however, t h a t a l t h o u g h these very high decay rates could a p p l y to the m a j o r i t y of splenic B cells, o t h e r B l y m p h o c y t e subsets m i g h t have c o n s i d e r a b l y longer life spans. These differences m a y result from r e g u l a t o r y m e c h a n i s m s o p e r a t i n g at the p e r i p h e r y a n d selecting for long life a few m e m b e r s o f the r a p i d l y t u r n i n g over o u t p u t from t h e



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marrow. This view might explain the wide discrepancies in the life span of B lymphocytes previously determined, which vary from 2 d to 7 wk, according to the experimental system and the clonal specificity analyzed (8-10). Summary T o determine the persistence of i m m u n o c o m p e t e n t B lymphocytes at the periphery, regardless of cell division, we have followed the decay o f lipopolysaccharide (LPS)reactive B cells in LPS-nonresponder, histocompatible hosts. Both the numbers of transferred cells and of those persisting in the recipients could be determined with precision by limiting dilution analysis o f the various cell populations. Decay rates of 50% per day were determined. Because we could exclude i m m u n e elimination of donor cells and the numbers of transferred cells were too low to result in gross alterations of the physiologic turnover rates, we conclude that the majority of LPSreactive B lymphocytes and a large part of the whole B cell c o m p a r t m e n t show this astonishingly high rate o f decay. T h e m e t h o d introduced here might prove useful in a variety of clonal assays, as it can detect cells present at a very low frequency. We thank C. H~ggstr/Jm for typing the manuscript, and M. Tuneskog for technical help.

Receivedfor publication 15June 1981. References I. Osmond, D. G., and G. J. V. Nossal. 1974. Differentiation of lymphocytes in mouse bonemarrow. II. Kinetics of maturation and renewal of antiglobulin-binding cells studied by double labeling. Cell ImmunoL 13: 132. 2. Andersson, J., A. Coutinho, W. Lernhardt, and F. Melchers. 1977. Clonal growth and maturation to immunoglobulin secretion "in vitro" of every growth-inducible B lymphocyte. Cell 10: 27. 3. Miller, R. G., and R. A. Philips. 1969. Separation of cells by velocity sedimentation.J. Cell Physiol. 73: 191. 4. Gronowicz, E., A. Coutinho, and F. Melchers. 1976. A plaque assay for all cells secreting Ig of a given type or class. Eur. J. ImmunoL 6: 588. 5. Freitas, A. A., and M. de Sousa. 1975. Control mechanisms of lymphocyte traffic. Modification of the traffic of 51Cr-labeled mouse lymph node cells by treatment with plant lectins in intact and splenectomized hosts. Eur. J. ImmunoL 5: 831. 6. Coutinho, A., and T. Meo. 1978. Genetic basis for unresponsiveness to lipopolysaccharide in C57BL/10.Cr mice. Immunogenetics. 7: 17. 7. Coutinho, A., L. Forni, and T. Watanabe. 1978. Genetic and functional characterization of an antiserum to the lipid A-specific triggering receptor on murine B-cells. Eur. J. ImmunoL 8: 63. 8. Coutinho, A., L. Forni, T. Watanabe, and F. Melchers. 1977. Genetic defect in responsiveness to LPS. Eur. J. ImmunoL 7: 325. 9. Strober, S. 1975. Immune function, cell surface characteristics and maturation of B cell subpopulations. Transplant. Rev. 24: 84. 10. Sprent, J. 1973. Circulating T and B lymphocytes in the mouse. Cell ImmunoL 7: 22. 11. Elson, C. J., K. F. Jablonska, and R. B. Taylor. 1976. Functional half-life of virgin and primed B lymphocytes. Eur. J. ImmunoL 6: 634.