Immunology Letters, 40 (1994) 89-92 0165 - 2478 / 94 / $ 7.00 © 1994 Elsevier Science B.V. All rights reserved

IMLET 02112

Research papers

Functional analysis of Mel- 14 + and Mel- 14-early precursor cells in the adult mouse thymus

Li Wu* and Angela D'Amico

The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia

(Received and accepted 15 February 1994)

1. Summary

The earliest T-cell precursor population in the adult mouse thymus (low CD4 precursors) may be divided into 85% of cells expressing surface Mel-14 (LECAM-1, the lymphocyte homing receptor) and 15% of cells which are Mel-14-. To date, this is the only surface marker for which we have found this po- pulation to be heterogeneous. The precursor activity of the Mel-14 ÷ and Mel-14- subpopulation was as- sessed by both intrathymic and intravenous transfer of sorted cells into Ly5 congenic irradiated recipient mice. On both a cell-for-cell and a total activity basis, almost all precursor activity was associated with the Mel-14 ÷ cells. No segregation was seen between T- cell, B-cell and dendritic cell precursor activity of the low CD4 population, all activities being concentrated in the Mel-14 ÷ fraction. This strengthens the hypoth- esis that one precursor cell has the potential to form all three lineages.

1. Introduction

Mel-14 (LECAM-1) is a lymphocyte homing re- ceptor. It is essential for the specific adherence of lymphocytes to high endothelial venules of the peri- pheral lymph nodes and their subsequent entry into the nodes [1-5]. Mel-14 is expressed on the thymic emigrants, on medullary CD4 ÷ 8 - and C D 4 - 8 ÷ thy- mocytes and some immature C D 4 - 8 - and CD4 +8 ÷ thymocytes [6,7]. The function of Mel-14 on these

Key words." T precursor; Lymphoid precursor; Mel-14

*Corresponding author: Li Wu, The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Vic- toria 3050, Australia.

immature thymocytes is not known. We now find that Mel-14 is expressed on some, but not all, of the earliest intrathymic precursors, the 'low CD4 precur- sors' we have previously isolated from the adult mouse thymus [8]. This population has precursor ac- tivity for T cells, B cells and dendritic cells (DC) [9,10]. Among many known surface markers 1 stu- died, Mel-14 is the first we have found to provide a clear division of this early precursor population. Here we assess the precursor activity of Mel-14 ÷ and Mel- 14- subpopulations.

2. Materials and Methods

The intrathymic low CD4 precursor cells were pre- pared using the procedures described elsewhere [8,9]. Briefly, thymocyte suspensions from 16 thymuses of 5- to 6-week-old C57BL/Ka-Thy-1.1-Ly5.2 mice were subjected to complement-mediated cytotoxicity and then immunomagnetic bead treatment to remove CD4 +8 ÷ thymocytes, mature CD3 ÷ thymocytes, the C D 4 - 8 - precursor cells, and non-T-lineage cells, all without the use of anti-CD4 antibody. The remaining cells were stained in three fluorescent col- ors with fluorescein isothiocyanate-conjugated-anti- Thy-l.1, phycoerythrin-conjugated-anti-HSA and biotin-anti-Mel-14 followed by Texas Red-avidin. The stained cells were sorted for cells expressing low levels of Thy-1, expressing moderate levels of heat- stable antigen (HSA), and which were positive or negative for Mel-14, as shown in Fig. 1. The sorted Mel-14 ÷ and Mel-14- precursor cells were then in- trathymically (i.t., 10,000/lobe) or intravenously (i.v.,

tThe markers tested were: CD2, CD3, CD4, CD8, Th-l, HSA, IL- 2R, CD44, MHC class I, MHC class I!, Ly6A/E, Sca-2, c-kit, B220 and NLDC-145.

SSDI 0 1 6 5 - 2 4 7 8 ( 9 4 ) 0 0 0 2 8 - P

1000

.,¢ 100

10

I I I

i0 i00 1000

Thy-1

" .. '

I I0 I00 1000

Mel-14

Fig. 1. Mel-14 distribution on the thymic low CD4 precursor po- pulation. Thymocyte suspensions were first depleted of more ma- ture thymocytes and non-T lineage cells, then the 1% remaining cells labeled and gated for cells which were low for Thy-1 and moderate for HSA (boxed area). The distribution of Mel-14 on these gated cells is shown in the lower histogram (solid line). The arrows in the histogram indicate the sorting gates used for Mel- 14 ~ and Mel-14- precursor cells. The dotted line represents the second stage background staining. Anti-CD4 was not used in the precursor isolation procedure.

30,000 cells/mouse) transferred into irradiated Ly5 congenic recipient mice. At various times after trans- fer, the donor-derived cells in the thymuses from i.t. injected mice, or donor-derived cells in the thymuses and in the spleen and lymph nodes from i.v. injected mice, were assessed by staining the cells with anti- Ly5.2 (donor type) together with anti-Thy-1 or anti- B220 or anti-Mac-I and anti-Gr-1, then analyzing with a flow cytometer, as described previously [9]. The lower number of donor-derived DC were as- sessed by using an DC enrichment procedure [10], and then staining the cells with anti-Ly5.2 (donor type) antibody together with anti-MHC class II and anti-NLDC-145, then analyzing by flow cytometry.

3. Results

Mel-14 is the only surface marker we have found

TABLE 1 T H Y M O C Y T E RECONSTITUT1ON AFTER 1NTRATHYMIC

INJECTION

Thymocyte re-population in the recipient mouse thymus 3 weeks after intrathymic transfer of 10,000 of each of the Mel-14 * and Mel-14- fractions of the low CD4 precursor cells. The precursor cells were obtained from C57BL Ly5.2 thymuses by depletion, 3- color fluorescent labeling and sorting as in Fig. 1. Three weeks after transfer into the irradiated Ly5.1 recipients, the recipient thymus cells were stained in three fluorescent colors for Ly5.2, CD4 and CD8, and the Ly5.2 + donor-derived cells analyzed. Two independent experiments yielded essentially the same results.

Donor-de}ived cells (%) Cells injected Donor-derived cells/lobe ( x l 0 '6) CD48 ~ CD4~8 CD4 8"

Mel-14 ~ precursors 5.4 82 14 3.5 Mel-14 precursors 1.0 83 12 4.5

so far which can subdivide the intrathymic low CD4 precursor population. As shown in Fig. 1, about 15- 20% of the precursor cells are Mel-14-, but the ma- jority are clearly Mel-14 ~. When the same number of Mel-14 + and Mel-14- precursor cells were trans- ferred i.t. into recipient mice, both subpopulations produced thymocyte progeny which were similar in terms of CD4 and CD8 expression (Table 1). How- ever, cell-for-cell the Mel-14 + precursors produced over 5-fold more progeny cells than did the Mel-14- precursors. Similar results were seen for thymic DC production (Table 2). Both subpopulations produced thymic DC, but on a cell-for-cell basis the Mel-14 ~ precursors produced about 10-fold more DC than the Mel- 14- precursors.

When transferred i.v., both Mel-14 + and Mel-14 subpopulations produced T cells in the recipient thy- mus and both subpopulations produced both T and

TABLE 2 T H Y M I C DENDRITIC CELL RECONSTITUTION AFTER 1NTRATHYMIC INJECTION

Thymic DC re-population in the recipient mouse thymus 2 weeks after intrathymic transfer of 9300 cells from Mel-14 + and Mel-14 fractions of the low CD4 precursor cells. The precursor cells were obtained from C57BL Ly5.2 mouse thymuses as in Fig. I. Two weeks after transfer into the irradiated Ly5.1 recipients, the DC were enriched from the recipient thymuses and stained in three fluorescent colors for Ly5.2, MHC class 11 and NLDC-145. The Ly5.2 + donor-derived DC were analyzed.

Cells injected Donor-derived Donor-derived T-linage cells/lobe DC/lobe ( x l O 6) ( x l O 3)

Mel-14 t precursors 5.1 4.4 Mel -14 precursors 0.7 0.4

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TABLE 3 RECONSTITUTION OF THYMUS, SPLEEN AND LYMPH NODES FOLLOWING INTRAVENOUS TRANSFER

Lymphoid lineage reconstitution in the recipient mouse thymus, spleen and lymph nodes (LN) 4 weeks after intravenous transfer of 34,000 cells from Mel-14 + and Mel-14 fractions of the low CD4 precursor cells. The precursor cells were obtained from C57BL Ly5.2 mice as in Fig. 1. Four weeks after intravenous transfer into the irradiated Ly5.1 recipients, the recipient thymus, spleen and lymph node cells were stained in two fluorescent colors for Ly5.2 and Thy-1, or B220, or Mac-1 and Gr-1, and the Ly5.2 ~ donor-derived cells analyzed. A second independent experiment gave similar results to those shown.

Cells injected Organ assayed Donor-derived cells/mouse ( x l 0 6)

Donor-derived cells (%)

Thy-I + B220 * M/G *~

Mel-14 + precursors Thymus 1.6 99 < 1 < 1 Spleen + LN 1.1 78 26 < 1

Mel-14 precursors Thymus 0.03 90 < 1 < 1 Spleen + LN 0.09 72 32 1.5

~M/G+: Mac-1 and Gr-1 positive cells.

B cells in the recipient spleen and lymph nodes (Table 3). The percentage of T- and B-cell production in the spleen and lymph node from the two precursor sub- populations was roughly the same. As shown pre- viously for the total low CD4 precursor population [9], no significant myeloid cell production was ob- tained with either subpopulation. In agreement with the results ofi.t , transfer, on i.v. transfer the Mel-14 ~ precursors also produced many more progeny cells than did the Mel-14 precursors (Table 3).

4. Discussion

Overall these results indicate that on a cell-for-cell basis, T-precursor, DC-precursor and potential B- precursor activity is much higher in the Mel-14 ~ sub- population of the low CD4 precursors. Since there were also many more Mel-14 + than Mel-14 precur- sors, most of the total precursor activity of the low CD4 population is within the Mel-14 + subpopula- tion. However, approximately 3% of the total precur- sor activity within the Mel-14- subpopulation ap- pears qualitatively similar to that in the Mel-14 ~ group.

Subdivision of the thymic low CD4 precursors on the basis of Mel-14 expression, the only marker so far for which they are clearly heterogeneous, has not seg- regated T-cell, B-cell or DC precursor activity. This strengthens considerably the hypothesis that this po- pulation consists of a common precursor for all three lineages. However, clonal analysis is still needed to prove this hypothesis.

The function of Mel-14 on these low CD4 precur- sor cells is not known, just as it is not clear for the intrathymic CD4 8- precurso'ts. It may be impor-

tant, along with CD44 [11], for seeding of their ear- lier bone marrow progenitors into the thymus. How- ever, the labeling of these cells with saturating amounts of anti-Mel-14 antibody has not reduced their ability to re-seed the thymus on i.v. transfer compared with that of non-Mel-14 fractionated cells (data not shown). Alternatively, Mel-14 may play a role in the interactions of developing cells with ele- ments of the surrounding thymic microenvironment.

Acknowledgements

This work was supported by the National Health and Medical Research Council, Australia. The authors would like to thank Dr. Katarina Lundberg for her help with i.t. injections, and Dr. Frank Bat- tye, Robyn Muir and Ralph Rossi for their expert assistance with flow cytometry analysis and sorting.

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