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Localization of PNA-binding and nonbinding thymus cells in peripheral lymphoid organs
CELLULAR
IMMUNOLOGY
Localization
60,228-233
(1981)
of PNA-Binding and Nonbinding Thymus Cells in Peripheral Lymphoid Organs
GEORG KRAAL,DAGMAR
BODEN,AND NEL KORS
Department of Histology, Medical Faculty, Free University, P.O. Box 7161. 1007 MC.Amsterdam, The Netherlands Received October 14. 1980; accepted December 29, 1980 Thymus cells were labeled in vitro with FITC and injected into syngeneic recipients. In cell suspensions of lymphoid organs green cells were inspected for PNA receptors with double immunofluorescence. A striking preference of PNA-negative cells to localize in lymph nodes and the lymphoid compartment of the spleen was demonstrated. Incubation with anti-Ly sera revealed that Ly l+ PNA-negative cells homed in popliteal lymph nodes and Peyer’s patch but not in mesenteric lymph nodes.
INTRODUCTION Surface receptors for peanut agglutinin (PNA), a lectin with D-g&iCtOSe specificity, are present on W-90% of thymocytes. The binding of the lectin does not lead to a mitogenic response and by sedimentation the agglutinated cells can be separated from the nonagglutinated (1). In combination with other surface markers such as alloantigens the separation with PNA has been used to delineate various thymocyte subpopulations (2, 3). Studies with fluorescent PNA revealed that the lectin also binds to a minority of peripheral lymphocytes which are either T cells or null cells (4, 5). During ontogeny PNA-positive cells can be detected in fetal liver and in embryonic thymus and spleen (6). Moreover, separation of PNA-positive cells from bone marrow and spleen showed an enrichment with hemopoietic cells in the positive fraction indicating that the receptor for PNA is a probable marker for immature cells (7). In the thymus the PNA-positive cells are thought to represent the immature cortical population, whereas the nonagglutinating cells represent the medullary immunocompetent fraction (1, 4). Labeling thymus cells with FITC in vitro which, when performed under physiological conditions does not influence their migration capacity or viability (8, 9), enabled us to study the homing patterns of these cells. After incubating organ suspensions with rhodaminated PNA the difference in migration pattern of PNAnegative and PNA-positive cells was investigated. A striking preference of PNAnegative cells for lymph nodes and the lymphoid compartment of the spleen was demonstrated. 228 0008-8749/81/070228-06$02.00/O Copyright 8 1981 by Academic Press, Inc. All rights of reproduction in any form rcscrwd.
229
SHORT COMMUNICATIONS
MATERIALS
AND
METHODS
Animals. CBA/J mice, 6 to 8 weeks old, were obtained from Zentralinstitut fur Versuchstierzucht, Hannover, Germany. DBA/2 mice were obtained from CPB, Zeist, The Netherlands. They were given acidified chlorinated water (pH 3) and commercial mouse food ad libitum. FZTC labeling and cell transfer. FITC (BBL, Isomer I) was dissolved in phosphate-buffered saline (PBS) (1 mg/ml) and frozen at -70°C in small aliquots. Thymus cell suspensions were labeled for 30 min at a concentration of 0.07 mg FITC/lO X lo6 cells/ml. The cells were washed three times in PBS and 50 X lo6 cells were injected intravenously. Before injection the suspensions were inspected for uniform staining. Two hours after injection of FITC-labeled thymus cells into syngeneic recipients organs were excised and single-cell suspensions were prepared by nylon gauze filtration. For cryostat sectioning organs were frozen in liquid nitrogen. Peanut agglutinin and antisera. Peanut agglutinin (PNA, IBF) was conjugated with tetramethylrhodamine isothiocyanate (TRITC Isomer R) (Nordic) at pH 9.5 for 90 min at a concentration of 16 mg PNA/mg TRITC. The conjugate was purified on a Sephadex G-75 column equilibrated with PBS (pH 7.4). Monoclonal anti-Ly 1 and anti-Ly 2 (53-7-213 and 53-6-72) were provided by Dr. van Ewijk. Their properties have been described elsewhere (10). Second-step rhodaminated anti-mouse IG was purchased from Nordic. RESULTS To determine whether PNA-positive and PNA-negative thymus cells exhibited different localization patterns in peripheral lymphoid organs the first experiments were set up with FITC-labeled thymus cells fractionated with PNA before labeling. However, in our hands these cells have a poor recovery and localization compared with unfractionated thymus cells (results not shown). Probably the separation procedure with PNA involving hypotonic galactose treatment and extensive washings after labeling made the cells too fragile to survive subsequent injection. For this reason we chose to demonstrate the PNA receptor after the donor cells had localized at various sites in the host organs. Localization
of PNA-Binding
and Nonbinding
Thymus
Ceils
After fixing the cell suspensions in Baker’s Form01 they were incubated with rhodaminated PNA. The number of FITC-labeled cells in the various organ suspensions was counted in the fluorescence microscope and by changing filters the percentages of green cells which also fluorescence with rhodamine were determined (Table 1). The thymus cell suspension which was injected, contained approximately 10% PNA-negative cells. Two hours after injection green FITC+ cells in comparable percentages of PNA-positive and PNA-negative cells as before injection were found in spleen and bone marrow, whereas a slight increase of cells that did not bear PNA receptors was found in the blood (Table 1). However, when the FITC cells that localized in lymph nodes and Peyer’s patches were inspected for PNA receptors a striking preference of PNA-negative cells for these organs was observed (Table 1).
230
SHORT COMMUNICATIONS TABLE
Organ
FITC-labeled cells (%)
Thymus cells injected Spleen Blood Bone marrow PLN MLN Peyer’s patches Thymus
1 FITC-labeled PNA-positive cells (%)
>99 l&12 1-2 3-4
90.2 88.4 73.1 83.7 12.9 10.5 8 -
FITC-labeled PNA-negative cells (%) 9.8 11.6 26.9 16.3 87.1 89.5 92 -
Note. CBA/J mice were injected iv with 50 X lo6 FITC-labeled thymus cells. Two hours later various organs were removed and suspensions were fixed in Baker’s Form01 and incubated with PNA-TRITC. In a fluorescence microscope (Leitz) the number of green cells was scored and by changing filters green cells with receptors for PNA were counted. Per organ at least 100 green cells were counted. The results are the mean of three separate experiments. PLN, Popliteal lymph node; MLN, mesenteric lymph node.
Spleen cryostat sections incubated with PNA-TRITC revealed that the PNAnegative cells were predominantly localized in the outer PALS of the white pulp (the spleen’s lymphoid compartment) whereas PNA-positive cells were found in the red pulp. Heat killing of the cells before injection (30 min, 56°C) changed the localization patterns dramatically, indicating that an active interaction of the cells and organs was necessary. Cryostat sections showed that dead, FITC cells were mainly found in the liver and spleen’s red pulp but particularly in the lungs, being the first capillary bed after tail vein injection. No FITC+ cells could be recovered from lymph nodes or bone marrow. The Influence of H-2 Differences on the Localization binding Cells
of PNA-Binding
and Non-
The same experiments were repeated with recipients with major H-2 differences from the donor. No quantitative differences in localization of FITC thymus cells could be observed in allogeneic recipients (Table 2). Again a predominant localization of PNA-negative cells in lymph nodes and especially in Peyer’s patches was observed. Lyt Phenotypes of FITS
Cells
Double immunofluorescence on thymus cells with PNA and Lyt 1 and Lyt 2 antisera showed that all PNA-positive cells reacted with Lyt 1 and Lyt 2 but that in the negative fraction a substantial population of Lyt 1+2- cells could be found (Table 3). Counterstaining of the FITC+ cells in the various organs 2 hr after injection revealed that in popliteal lymph node and Peyer’s patches approximately 25% of the FITC cells were Lyt 1+2- (Table 4). However, in the mesenteric node all FITC cells were Lyt 1+2+ which was also found for the FITC+ cells in blood, spleen, and bone marrow (Table 4).
231
SHORT COMMUNICATIONS TABLE
Organ
2 FlTC+-labeled PNA-positive cells (%)
FITC-labeled cells (%)
FITC’-labeled PNA-negative cells (96)
CBA + DBA Spleen MLN PLN PP
10
88.2 48.0 48.8 17.4
11.8 52.0 51.2 82.6
DBA - CBA Spleen MLN PLN PP
8
77.6 42.9 34.5 6.2
22.4 57.1 65.5 93.8
Nofe. CBA/J (H2k) and DBA/Z (H-2d) mice were injected with 50 X lo6 FITC-labeled thymus cells of the opposite strain. Two hours later various organs were removed and suspensions were fixed in Baker’s Formol and incubated with PNA-TRITC. In the fluorescence microscope (Leitz Orthoplan) the number of green cells was scored and by changing filters green cells with receptors for PNA were counted. At least 50 cells per organ were counted. PLN, Popliteal lymph node; MLN, mesenteric lymph node; PP. Peyer’s patch.
DISCUSSION Direct fluorescent labeling under physiological conditions of lymphocytes in vitro does not affect their viability nor migration capacity as has been extensively investigated by Butcher ef al. (8, 9). The selective migration as observed in our experiments must therefore reflect physiological migration properties of the fluorescent-labeled cells. Double immunofluorescence showed that approximately two-thirds of the PNAnegative cells are Ly l+ which is about 8% of the total thymus population, whereas one-third of the PNA-negative fraction is Ly 1+2+ (Table 3). Such a substantial number of Ly 1 cells in the thymus has also been reported by Mathieson (11) who showed that this population was increased after in vivo cortisone treatment. These findings indicate that the cortison-resistent medullary cells and the PNA-negative Ly l+ cells are the same population. The distribution of Lyt phenotypes in the PNA-negative thymus fraction is in accordance with the percentages found by Scollay et al. (12, 13). These authors studied thymus migrants after intrathymic cell labeling with FITC and showed that 67% of the thymus migrants are Ly l+ and 31% Ly 1+2+ (12). TABLE
3 Percentage
Thymus cells PNA-positive PNA-negative Nore. Using double-immunofluorescence antigens.
Anti-Ly >98 >98
1
Anti-Ly 2 >98 30
thymus cells were examined for PNA receptors and Ly
232
SHORT COMMUNICATIONS TABLE
4
Organs
FITC+ anti-Lyt 1
FITC+ anti-Lyt 2
Spleen Blood Bone marrow Popliteal lymph node Mesenteric lymph node Peyer’s patch
>98 >98 >98 >98 >98 >98
>98 >98 >98 76,8 >98 74,5
Note. CBA/J mice were injected iv with 50 X lo6 FITC-labeled thymus cells. Two hours later various organs were removed and single-cell suspensions were incubated with anti-Lyt 1 and anti-Lyt 2. After incubation with rhodaminated second-step antibodies the cells were examined in a Leitz fluorescence microscope. Per organ at least 100 cells were counted in two separate experiments.
The preferential homing of the PNA-negative cells in lymph nodes and Peyer’s patches which is abolished by cell death supports the concept that these PNAnegative cells are more immunocompetent and that cells that will leave the thymus for the periphery are among this fraction. Homing of lymphocytes in lymph nodes requires an active interaction of these cells with the endothelial cells of the high endothelial venules (HEV), which is the route by which blood-borne lymphocytes enter these organs (14). Differences in such interaction have been described by Butcher et al. who studied organ-specific lymphocyte migration by means of lymphocyte adherence to HEV (15). These authors have demonstrated the capacity of lymphocytes to distinguish between HEV of various lymph nodes and Peyer’s patches. Peyer’s patch HEV have a selective affinity for Peyer’s patch lymphocytes as is the case with nonmesenteric lymph node HEV for nonmesenteric lymph node lymphocytes. However, the mesenteric lymph node HEV holds an intermediate position by having almost equal affinity for Peyer’s patch and nonmesenteric node lymphocytes ( 15). In our experiments the FITC+ cells recovered from mesenteric lymph nodes are Ly 1+2+ in contrast to the popliteal lymph node and Peyer’s patch where many FITC LY I+ cells can be found. This also points to a different position of the mesenteric node among the HEVbearing organs. This organ selectivity must be based on receptor specificity of either endothelial cells and lymphocytes as has been outlined by Butcher et al. (15). Therefore, if we assume that the PNA-negative FITC cells found in these organs represent in part the physiological thymus migrants such receptor diversity must already have been developed in the thymus. ACKNOWLEDGMENTS The authors are indebted to Prof. Dr. H. L. Langevoort for his advice and criticism, to Dr. B. E. C. Plesch for correcting the English text, and to Dr. W. van Ewijk for the generous gift of the monoclonal antisera. We also thank Mrs. Anjo Steenvoorden-Bosma for typing the manuscript.
REFERENCES 1. Reisner, Y., Linker-Israeli, M., and Sharon, W. Cell. Immunol. 25, 129, 1976. 2. Imai, Y., Oguchi, T., Nakano, T., and Osawa, T. Immunol. Comm. 8, 5 11, 1979. 3. London. J.. and Horton. M. A. J. fmmunol. 124. 1803. 1980.
SHORT COMMUNICATIONS 4. 5. 6. 7. 8. 9. IO. Il. 12. 13. 14. 15.
233
London, J., Berrih, S., and Bach, J. F., J. Immunol. 121, 438, 1978. Roelants, G. E., London, J., Mayor-Withey, K. S., and Serrano, B., Eur. J. Immunol. 9, 139, 1979. London, J., Berrih, S., and Papiernik, M., Develop. Comp. Immunol. 3, 343, 1979. Reisner, Y., Itzicovitch, L., Meshorer, A., and Sharon, N., Proc. Nut. Acad. Sci. USA 75, 2933, 1979. Butcher, E. C., and Weissman, I. L., J. Immunol. Meth., 37, 97, 1980. Butcher, E. C., Scollay, R. Cl., and Weissman, I. L., J. Immunol. Merh., 37, 109, 1980. Ledbetter, J. A., and Herzenberg, L. A., Immunol. Rev. 47, 63, 1979. Mathieson, B. J., Sharrow, S. O., Campbell, P. S., and Asofsky, R., Nature (London) 277, 478, 1978. Scollay, R., Kochen, M., Butcher, E., and Weissman, I. L., Nufure (London) 276, 79, 1978. Scollay, R., and Weissman, I. L., J. Immunol. 124, 2841, 1980. Gowans, I. L., and Knight, E., Proc. Roy. Sot. Ser. E 159, 257, 1964. Butcher, E. C., Scollay, R. G., and Weissman, I. L., Eur. J. Immunol. 10, 556, 1980.
IMMUNOLOGY
Localization
60,228-233
(1981)
of PNA-Binding and Nonbinding Thymus Cells in Peripheral Lymphoid Organs
GEORG KRAAL,DAGMAR
BODEN,AND NEL KORS
Department of Histology, Medical Faculty, Free University, P.O. Box 7161. 1007 MC.Amsterdam, The Netherlands Received October 14. 1980; accepted December 29, 1980 Thymus cells were labeled in vitro with FITC and injected into syngeneic recipients. In cell suspensions of lymphoid organs green cells were inspected for PNA receptors with double immunofluorescence. A striking preference of PNA-negative cells to localize in lymph nodes and the lymphoid compartment of the spleen was demonstrated. Incubation with anti-Ly sera revealed that Ly l+ PNA-negative cells homed in popliteal lymph nodes and Peyer’s patch but not in mesenteric lymph nodes.
INTRODUCTION Surface receptors for peanut agglutinin (PNA), a lectin with D-g&iCtOSe specificity, are present on W-90% of thymocytes. The binding of the lectin does not lead to a mitogenic response and by sedimentation the agglutinated cells can be separated from the nonagglutinated (1). In combination with other surface markers such as alloantigens the separation with PNA has been used to delineate various thymocyte subpopulations (2, 3). Studies with fluorescent PNA revealed that the lectin also binds to a minority of peripheral lymphocytes which are either T cells or null cells (4, 5). During ontogeny PNA-positive cells can be detected in fetal liver and in embryonic thymus and spleen (6). Moreover, separation of PNA-positive cells from bone marrow and spleen showed an enrichment with hemopoietic cells in the positive fraction indicating that the receptor for PNA is a probable marker for immature cells (7). In the thymus the PNA-positive cells are thought to represent the immature cortical population, whereas the nonagglutinating cells represent the medullary immunocompetent fraction (1, 4). Labeling thymus cells with FITC in vitro which, when performed under physiological conditions does not influence their migration capacity or viability (8, 9), enabled us to study the homing patterns of these cells. After incubating organ suspensions with rhodaminated PNA the difference in migration pattern of PNAnegative and PNA-positive cells was investigated. A striking preference of PNAnegative cells for lymph nodes and the lymphoid compartment of the spleen was demonstrated. 228 0008-8749/81/070228-06$02.00/O Copyright 8 1981 by Academic Press, Inc. All rights of reproduction in any form rcscrwd.
229
SHORT COMMUNICATIONS
MATERIALS
AND
METHODS
Animals. CBA/J mice, 6 to 8 weeks old, were obtained from Zentralinstitut fur Versuchstierzucht, Hannover, Germany. DBA/2 mice were obtained from CPB, Zeist, The Netherlands. They were given acidified chlorinated water (pH 3) and commercial mouse food ad libitum. FZTC labeling and cell transfer. FITC (BBL, Isomer I) was dissolved in phosphate-buffered saline (PBS) (1 mg/ml) and frozen at -70°C in small aliquots. Thymus cell suspensions were labeled for 30 min at a concentration of 0.07 mg FITC/lO X lo6 cells/ml. The cells were washed three times in PBS and 50 X lo6 cells were injected intravenously. Before injection the suspensions were inspected for uniform staining. Two hours after injection of FITC-labeled thymus cells into syngeneic recipients organs were excised and single-cell suspensions were prepared by nylon gauze filtration. For cryostat sectioning organs were frozen in liquid nitrogen. Peanut agglutinin and antisera. Peanut agglutinin (PNA, IBF) was conjugated with tetramethylrhodamine isothiocyanate (TRITC Isomer R) (Nordic) at pH 9.5 for 90 min at a concentration of 16 mg PNA/mg TRITC. The conjugate was purified on a Sephadex G-75 column equilibrated with PBS (pH 7.4). Monoclonal anti-Ly 1 and anti-Ly 2 (53-7-213 and 53-6-72) were provided by Dr. van Ewijk. Their properties have been described elsewhere (10). Second-step rhodaminated anti-mouse IG was purchased from Nordic. RESULTS To determine whether PNA-positive and PNA-negative thymus cells exhibited different localization patterns in peripheral lymphoid organs the first experiments were set up with FITC-labeled thymus cells fractionated with PNA before labeling. However, in our hands these cells have a poor recovery and localization compared with unfractionated thymus cells (results not shown). Probably the separation procedure with PNA involving hypotonic galactose treatment and extensive washings after labeling made the cells too fragile to survive subsequent injection. For this reason we chose to demonstrate the PNA receptor after the donor cells had localized at various sites in the host organs. Localization
of PNA-Binding
and Nonbinding
Thymus
Ceils
After fixing the cell suspensions in Baker’s Form01 they were incubated with rhodaminated PNA. The number of FITC-labeled cells in the various organ suspensions was counted in the fluorescence microscope and by changing filters the percentages of green cells which also fluorescence with rhodamine were determined (Table 1). The thymus cell suspension which was injected, contained approximately 10% PNA-negative cells. Two hours after injection green FITC+ cells in comparable percentages of PNA-positive and PNA-negative cells as before injection were found in spleen and bone marrow, whereas a slight increase of cells that did not bear PNA receptors was found in the blood (Table 1). However, when the FITC cells that localized in lymph nodes and Peyer’s patches were inspected for PNA receptors a striking preference of PNA-negative cells for these organs was observed (Table 1).
230
SHORT COMMUNICATIONS TABLE
Organ
FITC-labeled cells (%)
Thymus cells injected Spleen Blood Bone marrow PLN MLN Peyer’s patches Thymus
1 FITC-labeled PNA-positive cells (%)
>99 l&12 1-2 3-4
90.2 88.4 73.1 83.7 12.9 10.5 8 -
FITC-labeled PNA-negative cells (%) 9.8 11.6 26.9 16.3 87.1 89.5 92 -
Note. CBA/J mice were injected iv with 50 X lo6 FITC-labeled thymus cells. Two hours later various organs were removed and suspensions were fixed in Baker’s Form01 and incubated with PNA-TRITC. In a fluorescence microscope (Leitz) the number of green cells was scored and by changing filters green cells with receptors for PNA were counted. Per organ at least 100 green cells were counted. The results are the mean of three separate experiments. PLN, Popliteal lymph node; MLN, mesenteric lymph node.
Spleen cryostat sections incubated with PNA-TRITC revealed that the PNAnegative cells were predominantly localized in the outer PALS of the white pulp (the spleen’s lymphoid compartment) whereas PNA-positive cells were found in the red pulp. Heat killing of the cells before injection (30 min, 56°C) changed the localization patterns dramatically, indicating that an active interaction of the cells and organs was necessary. Cryostat sections showed that dead, FITC cells were mainly found in the liver and spleen’s red pulp but particularly in the lungs, being the first capillary bed after tail vein injection. No FITC+ cells could be recovered from lymph nodes or bone marrow. The Influence of H-2 Differences on the Localization binding Cells
of PNA-Binding
and Non-
The same experiments were repeated with recipients with major H-2 differences from the donor. No quantitative differences in localization of FITC thymus cells could be observed in allogeneic recipients (Table 2). Again a predominant localization of PNA-negative cells in lymph nodes and especially in Peyer’s patches was observed. Lyt Phenotypes of FITS
Cells
Double immunofluorescence on thymus cells with PNA and Lyt 1 and Lyt 2 antisera showed that all PNA-positive cells reacted with Lyt 1 and Lyt 2 but that in the negative fraction a substantial population of Lyt 1+2- cells could be found (Table 3). Counterstaining of the FITC+ cells in the various organs 2 hr after injection revealed that in popliteal lymph node and Peyer’s patches approximately 25% of the FITC cells were Lyt 1+2- (Table 4). However, in the mesenteric node all FITC cells were Lyt 1+2+ which was also found for the FITC+ cells in blood, spleen, and bone marrow (Table 4).
231
SHORT COMMUNICATIONS TABLE
Organ
2 FlTC+-labeled PNA-positive cells (%)
FITC-labeled cells (%)
FITC’-labeled PNA-negative cells (96)
CBA + DBA Spleen MLN PLN PP
10
88.2 48.0 48.8 17.4
11.8 52.0 51.2 82.6
DBA - CBA Spleen MLN PLN PP
8
77.6 42.9 34.5 6.2
22.4 57.1 65.5 93.8
Nofe. CBA/J (H2k) and DBA/Z (H-2d) mice were injected with 50 X lo6 FITC-labeled thymus cells of the opposite strain. Two hours later various organs were removed and suspensions were fixed in Baker’s Formol and incubated with PNA-TRITC. In the fluorescence microscope (Leitz Orthoplan) the number of green cells was scored and by changing filters green cells with receptors for PNA were counted. At least 50 cells per organ were counted. PLN, Popliteal lymph node; MLN, mesenteric lymph node; PP. Peyer’s patch.
DISCUSSION Direct fluorescent labeling under physiological conditions of lymphocytes in vitro does not affect their viability nor migration capacity as has been extensively investigated by Butcher ef al. (8, 9). The selective migration as observed in our experiments must therefore reflect physiological migration properties of the fluorescent-labeled cells. Double immunofluorescence showed that approximately two-thirds of the PNAnegative cells are Ly l+ which is about 8% of the total thymus population, whereas one-third of the PNA-negative fraction is Ly 1+2+ (Table 3). Such a substantial number of Ly 1 cells in the thymus has also been reported by Mathieson (11) who showed that this population was increased after in vivo cortisone treatment. These findings indicate that the cortison-resistent medullary cells and the PNA-negative Ly l+ cells are the same population. The distribution of Lyt phenotypes in the PNA-negative thymus fraction is in accordance with the percentages found by Scollay et al. (12, 13). These authors studied thymus migrants after intrathymic cell labeling with FITC and showed that 67% of the thymus migrants are Ly l+ and 31% Ly 1+2+ (12). TABLE
3 Percentage
Thymus cells PNA-positive PNA-negative Nore. Using double-immunofluorescence antigens.
Anti-Ly >98 >98
1
Anti-Ly 2 >98 30
thymus cells were examined for PNA receptors and Ly
232
SHORT COMMUNICATIONS TABLE
4
Organs
FITC+ anti-Lyt 1
FITC+ anti-Lyt 2
Spleen Blood Bone marrow Popliteal lymph node Mesenteric lymph node Peyer’s patch
>98 >98 >98 >98 >98 >98
>98 >98 >98 76,8 >98 74,5
Note. CBA/J mice were injected iv with 50 X lo6 FITC-labeled thymus cells. Two hours later various organs were removed and single-cell suspensions were incubated with anti-Lyt 1 and anti-Lyt 2. After incubation with rhodaminated second-step antibodies the cells were examined in a Leitz fluorescence microscope. Per organ at least 100 cells were counted in two separate experiments.
The preferential homing of the PNA-negative cells in lymph nodes and Peyer’s patches which is abolished by cell death supports the concept that these PNAnegative cells are more immunocompetent and that cells that will leave the thymus for the periphery are among this fraction. Homing of lymphocytes in lymph nodes requires an active interaction of these cells with the endothelial cells of the high endothelial venules (HEV), which is the route by which blood-borne lymphocytes enter these organs (14). Differences in such interaction have been described by Butcher et al. who studied organ-specific lymphocyte migration by means of lymphocyte adherence to HEV (15). These authors have demonstrated the capacity of lymphocytes to distinguish between HEV of various lymph nodes and Peyer’s patches. Peyer’s patch HEV have a selective affinity for Peyer’s patch lymphocytes as is the case with nonmesenteric lymph node HEV for nonmesenteric lymph node lymphocytes. However, the mesenteric lymph node HEV holds an intermediate position by having almost equal affinity for Peyer’s patch and nonmesenteric node lymphocytes ( 15). In our experiments the FITC+ cells recovered from mesenteric lymph nodes are Ly 1+2+ in contrast to the popliteal lymph node and Peyer’s patch where many FITC LY I+ cells can be found. This also points to a different position of the mesenteric node among the HEVbearing organs. This organ selectivity must be based on receptor specificity of either endothelial cells and lymphocytes as has been outlined by Butcher et al. (15). Therefore, if we assume that the PNA-negative FITC cells found in these organs represent in part the physiological thymus migrants such receptor diversity must already have been developed in the thymus. ACKNOWLEDGMENTS The authors are indebted to Prof. Dr. H. L. Langevoort for his advice and criticism, to Dr. B. E. C. Plesch for correcting the English text, and to Dr. W. van Ewijk for the generous gift of the monoclonal antisera. We also thank Mrs. Anjo Steenvoorden-Bosma for typing the manuscript.
REFERENCES 1. Reisner, Y., Linker-Israeli, M., and Sharon, W. Cell. Immunol. 25, 129, 1976. 2. Imai, Y., Oguchi, T., Nakano, T., and Osawa, T. Immunol. Comm. 8, 5 11, 1979. 3. London. J.. and Horton. M. A. J. fmmunol. 124. 1803. 1980.
SHORT COMMUNICATIONS 4. 5. 6. 7. 8. 9. IO. Il. 12. 13. 14. 15.
233
London, J., Berrih, S., and Bach, J. F., J. Immunol. 121, 438, 1978. Roelants, G. E., London, J., Mayor-Withey, K. S., and Serrano, B., Eur. J. Immunol. 9, 139, 1979. London, J., Berrih, S., and Papiernik, M., Develop. Comp. Immunol. 3, 343, 1979. Reisner, Y., Itzicovitch, L., Meshorer, A., and Sharon, N., Proc. Nut. Acad. Sci. USA 75, 2933, 1979. Butcher, E. C., and Weissman, I. L., J. Immunol. Meth., 37, 97, 1980. Butcher, E. C., Scollay, R. Cl., and Weissman, I. L., J. Immunol. Merh., 37, 109, 1980. Ledbetter, J. A., and Herzenberg, L. A., Immunol. Rev. 47, 63, 1979. Mathieson, B. J., Sharrow, S. O., Campbell, P. S., and Asofsky, R., Nature (London) 277, 478, 1978. Scollay, R., Kochen, M., Butcher, E., and Weissman, I. L., Nufure (London) 276, 79, 1978. Scollay, R., and Weissman, I. L., J. Immunol. 124, 2841, 1980. Gowans, I. L., and Knight, E., Proc. Roy. Sot. Ser. E 159, 257, 1964. Butcher, E. C., Scollay, R. G., and Weissman, I. L., Eur. J. Immunol. 10, 556, 1980.