CD45 up-regulation during lymphocyte maturation

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1 Internatonal Immunology, Vol., No. 11, pp Oxford Unversty Press CD45 up-regulaton durng lymphocyte maturaton Jorg Krberg and Thomas Brocker Basel Insttute for Immunology, Grenzacherstrasse 47, Postfach, 45 Basel, Swtzerland Keywords: CD45 up-regulaton, mouse, postve selecton, sgnalng molecules, T lymphocytes Abstract CD4 + + double-postve thymocytes dfferentate nto CD4 + and + sngle-postve T cells durng thymc postve selecton. Ths process requres the nteracton between the TCR and self MHC molecules. In ths context we have analyzed the expresson of CD45, an abundant transmembrane proten tyrosne phosphatase, and descrbe here ts dfferental surface expresson durng T cell maturaton. Usng four-color FACS analyss of thymocytes from normal as well as TCR-transgenc mce we demonstrate that CD45 s up-regulated only durng postve selecton concomtantly wth the TCR-CD3 complex and the transent early actvaton marker CD69, but that ths up-regulaton precedes heat stable antgen down-regulaton. The tght lnkage of the upregulaton of the TCR-CD3 complex and CD45 may be requred because the CD45 tyrosne phosphatase plays a role n modulatng sgnal transducton by the TCR-CD3 complex durng postve selecton. In addton, our fndngs argue for a regulaton mechansm that adapts the CD45 levels to ncreasng antgen receptor levels on mature T cells and B cells. Introducton CD45 s a transmembrane proten tyrosne phosphatase abundantly present on the cell surface of all nucleated hematopoetc cells. Ths proten exsts n several soforms due to alternatve splcng of four of ts exons (for revews see 1,2). Studes wth CD45" mutant cell lnes that were antgen receptor "sgnalng defcent showed that expresson of CD45 s a prerequste for transmembrane sgnal transducton upon trggerng of the TCR (3,4) as well as the B cell antgen receptor (5). It was further demonstrated that CD45 dephosphorylates specfcally negatve regulatory tyrosne resdues of src famly proten tyrosne knases, an event that results n ncreased knase actvty n T cells (6-9) and B cells (5,1,11). The dsrupton of the CD45 gene underlned clearly the essental role of ths phosphatase n T lymphocyte development and B cell functon, snce t resulted n a drastc reducton of mature sngle-postve thymocyte numbers and mature B lymphocytes wth sgnalng-defcent antgen receptors (12). Proten phosphorylaton s an mportant mechansm of transducng sgnals n eukaryotc cells. It has been proposed that a restng cell s mantaned below a threshold for sgnalng through a balance between opposng dephosphorylaton and phosphorylaton reactons (13). In fact, chemcal nactvaton of phosphatases n Jurkat cells leads to IL-2 producton wthout TCR trggerng (14), whch ndcates that the cell has to regulate both opposng actvtes tghtly to keep them n balance. Lttle s yet known about the regulaton of CD45-phosphatase actvty. It has been suggested that the dfferent soforms of CD45 could nteract wth dfferent lgands and become thereby dfferentally actvated (for revew see 2). When mouse thymocytes were prevously nvestgated for expresson of certan soforms that mght correlate to postve or negatve selecton events, not all analyzed thymocytes (only 1-3%) expressed specfc soforms (15). Therefore, n the present study we used three dfferent CD45 pan-soform-specfc reagents together wth CD4, and CD3, heat stable antgen (HSA) or CD69 as thymocyte markers as well as IgM, IgD, CD19 or B22 as B lymphocyte markers and extensvely analyzed CD45 expresson (regardless of soforms) n varous thymocyte subsets as well as durng B cell development. In clear contrast to earler reports (16), we demonstrate n ths study that there s an extremely tght correlaton between CD45 expresson levels and postve selecton events n the thymus. More than 9% of all postvely selected thymocytes showed a CD45 hqh phenotype, whereas bascally all nonselected thymocytes staned CD45 OW. Smlarly, we could detect a drastc CD45 up-regulaton durng B cell development that paralleled B cell antgen receptor up-regulaton and B Correspondence to: T. Brocker Transmttng edtor. B. Malssen Receved 25 March 1996, accepted 27 July 1996

2 1744 CD45 up-regulaton and thymc postve selecton Q O -..'....- \ CO o 29 t o T,fc "I* r CD45(Ly5 a ) Fg. 1. Four-color FACS analyss of B6.SJL (Ly-5 a ) thymocytes: CD4 and expresson was analyzed wth a gate set on forward versus sde scatter parameters to exclude dead cells and debrs. Subsequently, nne gates, correspondng to dfferent developmental stages between CD4 + + DP and CD4 + SP and + SP thymocytes, were ntroduced. The lower three panels show for each of these nne gates the analyss wth the followng pars of mab: lower left, center and rght panels show CD45 (clone ) versus CD3, CD69 or HSA respectvely. In each panel, gate 1 (CD4 + + DP cells) corresponds to the top mddle poston, whle gates 2-5 descend to the left (towards CD4 + SP cells) and gates 6-9 descend to the rght (towards + SP cells). A total of 3X1 5 cells were analyzed. All cells wthn the forward versus sde scatter parameters are dsplayed n the top panel, whereas 2 cells of the gated subpopulatons are shown n the small panels below.

3 CD45 up-regulaton and thymc postve selecton 1745 Table 1. CD45 and TCR up-regulaton durng thymocyte maturaton Total Gate 1 CD4++ Gate 2 Gate 3 Gate 4 Gate 5 Gate 6 CD4+- CD45 CD3 CD Data are derved from the same analyss as shown n Fg. 1. Smlar to Fg. 1, sx gates were set to cover the developmental transton between mmature CD4 + + and mature CD4 + ~ T cells. The mean relatve fluorescence ntensty of CD45, CD3 and CD4 of the varous subpopulatons s shown. cell maturaton. We dscuss the mplcatons of these fndngs wth respect to TCR and B cell receptor expresson levels durng lymphocyte development. Methods Mce B6.SJL (Ly-5 a ) and C56BL/6 mce were purchased from Jackson Laboratores (Bar Harbor, ME) or IFFA Credo (Orleans, France) respectvely. TCR transgenc mce, wth a hemagglutnn-specfc, l-e d -restrcted TCR, were as descrbed before and crossed to a recombnaton actvatng gene negatve (RAG-2^-) background (17). Snce RAG-2"'" mce were a mxture of Ly-5 a and Ly-5 b mce, we selected for these markers to obtan Ly-5 a or Ly-5 b homozygotes. Phenotypng was done by FACS stanng of perpheral blood lymphocytes wth mab specfc for TCR Vp, TCR clonotype, Ly-5 allele and D d or K b. Mce were bred n the anmal colony of the Basel Insttute for Immunology and were analyzed at 6-1 weeks of age. mab and FACS analyss Hybrdoma supernatants contanng mab were purfed by Proten G (Pharmaca, San Dego, CA)-affnty chromatography. mab were labeled usng botn- or fluorescensuccnmdyl-ester (FLUOS) accordng to the manufacturer's nstructons. The followng mab were used: T (ant- D d ) (17), AF (ant-k b ) (1), M1/69 (ant-hsa) (19), KT-3 (ant-cd3) (2), F23.1 (ant-tcr V p ) (21), A2-1.7 (ant- Ly-5 b ) (22), (ant-ly-5 a ) (22) and 6.5 (ant-clonotypc TCR) (23). Ant-CD4-phycoerythrn (Becton Dcknson, Plymouth, UK), ant--red613 (Gbco/lmmunoselect/BRL, Grand Island, NY), ant-lgd (clone ; PharMngen, San Dego, CA), ant-igm (clone AF6-7; PharMngen), RA3-6B2 (ant-cd45, B22 soform; PharMngen) and ant-cd45-fitc (clone 3F11.1, specfc for all CD45 soforms; PharMngen) conjugates were obtaned commercally. Wth these and the second step reagent streptavdn-allophycocyann (APC; Molecular Probes, Eugene, OR) four color flow-cytometry was performed on a FACStar" 1 " (Becton Dcknson) nstrument equpped approprately. Sngle cell preparaton, stanng and FACS analyss were done accordng to standard procedures. Results In order to nvestgate the expresson of CD45 on thymocytes of B6.SJL mce that were homozygous for one of the CD45 alleles (Ly-5 a ), we performed four-color flow cytometrc analyss wth mab specfc for CD4,, CD45 and a fourth marker (CD3, CD69 or HSA respectvely) (Fg. 1). We frst analyzed the thymocytes accordng to ther CD4 and expresson and gated them n dstnct developmental stages between double-postve (DP) and sngle-postve (SP) thymocytes (see Fg. 1, gates 1-9). We further analyzed the cells n gates 1-9 accordng to ther expresson of CD45 versus ether CD3, CD69 or HSA. Fgure 1 shows that the expresson levels of CD45 ncrease durng maturaton of thymocytes (Fg. 1, lower part, wth the DP fracton of gate 1 on the top of the two columns and the maturng CD4 + SP cells shown n the dot-plots descendng to the left, the + SP cells to the rght). In the lower left panel of Fg. 1, CD45 expresson s correlated wth TCR-CD3 complex expresson as measured by a mab specfc for CD3e. Whle all DP thymocytes are dull for CD3 and postve for CD45, >9% of the most mature CD4 + SP cells are expressng hgher levels of CD3 and CD45. The same shft can be observed for + SP thymocytes wth the remanng CD3 dull CD45 low cells beng mmature + SP (24,25). When we compared CD69 and CD45 (Fg. 1) we observed the expected transent up-regulaton of CD69 (26) by thymocytes on ther way to mature CD4 + SP or + SP cells. Ths transent up-regulaton of CD69 takes place at the same stage as CD45 up-regulaton. Wth further maturaton CD69 s downregulated agan, whle CD45 expresson levels reman hgh (Fg. 1, gates 5 and 9). Immature + SP thymocytes reman CD45 low (see lower rght dot-plot correspondng to gate 9). We then analyzed the expresson of CD45 n the context of HSA. As descrbed prevously, mmature thymocytes express hgh levels of HSA and lose ths marker on ther way to mature SP cells (27). Accordng to Fg. 1 (rght lower panel) practcally all of the more mature thymocytes became CD45 h ' 9h before they started to lose HSA expresson. Quanttatve analyss of three surface markers shows that CD4 levels do not change, CD3 s up-regulated -12 tmes and CD45 levels are ncreased 2-fold durng thymocyte maturaton as shown n Table 1. In order to see whether ths event s dependent on postve selecton, we repeated the experments n RAG-2~'~ TCRtransgenc mce that can or cannot postvely select mmature thymocytes. As prevously descrbed (17), n the I-E d+ TCRtransgenc RAG-2"'" mce, postve selecton leads to the presence of CD4 + SP and + SP mature thymocytes (Fg. 2) whle n the correspondng -E 6 ' mce such cells cannot

4 1746 CD45 up-regulaton and thymc postve selecton 1-! 2 2 D O a o " IF 3 * lfc.>.-' ' '* 3 ^91 1 *"* '*' * lfc> ' " " *99.1 : I" - 9 I '24 I 1! 76 1 tt> 1*' f I** 1* t I " s * r " "11 V 1 47' 1 I 1 J r I L "A- 173 J 1u 95 1 :. *4 f f '2 a-.» t CD45(Ly5 a ) 97 I 1 '2 t#* < '!«!» :*-

5 CD45 up-regulaton and thymc postve selecton + + be detected (Fg. 2). We gated on CD4 DP, CD4+ SP or + SP thymocytes (gates 1, 2 and 3), and analyzed them for CD45 versus CD3, CD69 and HSA. In case of I-Ed+ TCR-transgenc RAG-2"'"" mce we observed the same correlaton between these markers as descrbed above: CD45 s up-regulated durng postve selecton on thymocytes (Fg. 2). In contrast, thymocytes from I-Ed"TCR-transgenc RAG-2^" mce that cannot postvely select wth ths TCR reman CD45lW (Fg. 2). Thus CD45 up-regulaton s dependent on postve selecton events. To test whether CD45 up-regulaton s allele-specfc we compared I-Ed+ and I-Ed" TCR-transgenc RAG-2~'~ mce that were homozygous for the CD45 allele Ly-5b. As shown n Fg. 3, we obtaned smlar results as n Ly-5a mce. Apparently CD45 up-regulaton upon postve selecton s a general phenomenon, not restrcted to a partcular allele. Whle the majorty of thymocytes and actvated T cells express dfferent low mol. wt soforms of CD45, B22, a hgh molecular soform of CD45, has been routnely used as a marker for all B lneage cells (1,2). We were nterested f the observed CD45 up-regulaton durng T cell maturaton could smlarly be detected wth a pan-soform CD45 specfc mab on developng B cells n the bone marrow. To ths end we analyzed bone marrow cells as shown n Fg. 4. To concentrate on B lneage cells, we frst gated on B22 + cells (data not shown). Accordng to ther surface IgM and IgD expresson we dvded these nto four stages correspondng to gates 1-4: IgM/lgD double negatve (gate 1), lgmlw SP (gate 2), gmhgh S P a n d gmhgh/ gd' w DP (gate3), and IgM/lgD DP (gate 4), reflectng the normal B cell developmental pathway. When these four populatons were analyzed for CD45 expresson levels, we found an up-regulaton of CD45 (see hstograms n Fg. 4) tghtly lnked to surface Ig up-regulaton and B cell maturaton. / CD < * * j. CD4 3 j CD4+- CD4 + ~ CD4+-* CD J v CD4~+ CD4S+ >,1 CD45(Ly5 b ) Fg. 3. Three-color FACS analyss of I-E+ and I-E" RAG-2~'~ Ly-5b TCR-transgenc thymocytes. Analyss was performed as descrbed for Fg. 2, but wth a pan-specfc CD45 mab (clone 3F11.1). Fg. 2. Four-color FACS analyss of I-Ed+ and I-Ed~ RAG-2^" Ly-5a TCR-transgenc thymocytes. Analyss and data dsplay was performed as descrbed for Fg. 1. CD4 + + DP, CD4 + SP or + SP thymocytes were analyzed usng gates 1, 2 and 3 as shown. In each panel, CD4 + SP, CD4 + + DP and + SP cells correspond to the upper left, upper rght or lower rght dot-plot respectvely.

6 174 CD45 up-regulaton and thymc postve selecton BM a. -' -j. ^-f a- Q TT>3 IgM CD45 Fg. 4. Four-color FACS analyss of bone marrow. Bone marrow cells were solated from C57BL/6 mce. Dead cells and debrs was excluded wth a gate set on forward versus sde scatter parameters. For further analyss, only B22 + cells were ncluded. These were dvded nto four populatons based on IgM and IgD expresson. For each of these four populatons, the hstograms show the expresson of CD45 (clone 3F11.1). Dscusson Earler studes have shown that t s dffcult to defne rules for the regulaton of CD45 soform expresson on thymocytes, snce only mnmal populatons (1-3%) express specfc soforms (CD45RA and CD45RBh'9h) that occur durng postve or negatve selecton n the thymus (15). Snce a much hgher percentage of thymocytes are postvely selected n normal and TCR-transgenc mce but do not express or up-regulate certan CD45 soforms, we nvestgated the overall expresson levels of CD45 on thymocytes wth pan-cd45 antbodes to see f soform-ndependent regulaton mght occur. Usng fourcolor flow cytometry we analyzed thymocytes from normal and TCR-transgenc mce and compared CD45 expresson levels wth other more commonly used thymocyte markers lke CD3, CD69 and HSA. As measured wth three dfferent mab specfc for all soforms of CD45, we descrbe here that CD45 expresson levels are lower on CD4 + + DP thymocytes, but ncrease when these cells mature to the CD4+ SP or + SP stage. The CD45 up-regulaton occurs smultaneously wth an ncrease of TCR-CD3 levels and one mght argue that the CD45 phosphatase levels are ncreased because of an ongong ncrease of TCR-CD3 levels n order to regulate sgnals receved durng thymocyte postve selecton. Nearly all (>9%) thymocytes followed ths rule. Interestngly, durng B cell development, the CD45 soform B22 shows a smlar shft n surface expresson (29). However, as yet undetected was the general, soform-ndependent up- regulaton of CD45 expresson when B cells start to express Ig, as shown n ths study. These fndngs ndcate that the total CD45 levels (regardless of ts soforms) may be mportant n developng lymphocytes to regulate sgnal transducton actvty durng lymphocyte maturaton. Acknowledgements The authors would lke to thank Mark Dessng for expert techncal assstance wth FACS analyss, Hanspeter Stahlberger for graphc art work, and Drs Kerry Campbell, Hans-Remer Rodewald, Klaus Karjalanen and Harald von Boehmer for readng the manuscrpt. The mab A2-1.7 and were a knd gft of Dr Hans-Remer Rodewald. The Basel Insttute for Immunology was founded and s supported by Hoffmann-La Roche Ltd, Basel, Swtzerland. Abbrevatons APC DP HSA SP allophycocyann double postve heat shock antgen sngle postve References 1 Thomas, M. L The leukocyte common antgen famly. Annu. Rev. Immunol. 7: Trowbrdge, I. S. and Thomas, M. L CD45: an emergng

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