The T-Cell Receptor V8 Genes Predominantly Used by Human Peripheral 3,13 + T Lymphocytes Are Not Rearranged in CD3- Natural Killer Cells

Sergio Roman-Roman, Elena Baixeras, Catherine Genevde, Thierry Hercend, and Fr6d6ric Triebel

ABSTRACT: We hare analyzed, in 19 CD3 natural killer cell clones, the genomic organization o/the T-cell receptor 3 locus wi th two distinct V8 probes, VSI and V82. These two V8 genes code for surface proteins expressed in more than 90% of peripheral blood T-cell receptor y /8 ~ lympho~ytes, as shown by double color immunofluorescence analysis with ant i -TCRS1, anti-BB3, and anti-STCS1 monoclonal antibodies. The VSI and V82 genes were found to be in germline position in all these clones, which are distinct phenotypically and represent a variety of the corresponding peripheral natural killer cell populations. We also studied in these cloned cell lines the transcriptional activity of the T-cell receptor 8 locus with a C8 probe: short transcripts (I. 7 and 0.8 kb) were found exclusively. These experiments further suggest that CD3 natural killer peripheral cells are likely to constitute a unique lineage distinct from T lymphocytes.

A B B R E V I A T I O N S C constant NK natural killer D diversity PBL peripheral bh)od lymphocytes J joining TcR T-cell receptor MoAb monoclonal antibody V variable

I N T R O D U C T I O N

Human peripheral natural killer (NK) cells are CD3 NKH1 ~ lymphocytes which can mediate cytotoxic reactions without apparent presensitization anti without restriction by the expression of class I or class II major histocompatibility antigens on target cells [1]. The N K cell fraction, which represents approxi- mately 10% of peripheral blood lymphocytes (PBL), is heterogeneous in the expression of cell surface antigens such as CD2, CD8, or N K H 2 [2]; moreover, individual N K cells are distinct in their ability to kill different target cells [3]. The cellular lineage of the CD3- N K cells as well as their recognition structures are

From the Laboratoire d'Immunologie Cellulaire, Ddpartement de Biologie Clinique. lnstitut Gastare- Rous.(y. Villejuif, France.

Address reprint requests to Dr. F. Triebel. Laboratoire d'tmmunologie Cel/ulaire. Dipartement de Biologic Chnique, Institut Gustat,e-Roussy, Villejuif. France.

Receired October 1 I, I988; ret, ised March 31. 1989.

Human Immunology 26, 75-83 (1989) 75 ~-~ American Society for Histocompatibility and Immunogenetics, 1989 0198-8859/89/$3.50

76 F. Triebel crai

still unknown. Studies on the genomic organization as well as the transcriptio~ial activity of the T-cell receptor (TcR) /3 and y genes have been performt, d previously in an at tempt to assess potential relationships between N K and T lymphocytes. D N A s extracted from the whole N K cell fraction in peripherai blood were found to be in germline configuration for TcR/3 and T c R y genes [4,5]. Given the heterogeneity of N K cells, these studies could have potentiall} missed specific rearrangements present in distinct C D 3 N K H I ~ subpopts lations. Thus, we have analyzed previously [6] the genomic organization of TcR/:~ and T c R y genes in a panel of 14 well-characterized N K clones that wt, re selected to represent, at least in part, the phenotypic heterogeneity (i.t:., C D 2 CD8 ~'~-, N K H 2 ' ) of circulating CD3 N K H I ~ lymphocytes. These c'xpt*ri ments showed that all N K clones tested were in germline configuration fi~r boti, the TcR/3 and T c R y loci.

Thus far, four variable (V) genes have been shown to rearrange at the 6 loctis One belongs to the V~6 family [7], indicating that, in rare cases, functional i~ chains can be created in humans by the rearrangement of V~ genes to the 6 locus The first human V8 gene that has been characterized (termed here "VSl") wai:, found in two different unrelated cell lines, IDP2 [8] and PEER [9], and thc~n it, ~ series of normal or leukemic cells [10]. More recently, we have identified ~ti additional V8 segment, V82 in a TiTA ~ cloned cell line, termed AB 12, deriw:d from normal peripheral blood [ l I ]. An identical sequence has been reported b', Hata et al. [ 12] and Loh et al. [13]. Finally, one V8 gene lies in an invertc.~i orientation 3' of constant (c) 8 [12,1;i].

The VS1 segment codes for a 8 protein which is recognized most likely [ 15 } b', the anti-STCS1 [16, 17] monoclonal antibody (MoAb). This V8 protein is surface-expressed in a minor but substantial fraction of the CD3 ~ TcR r/8 peripheral blood cell population [15] delineated by the pan-8 MoAb anti-TcR 6! [18]. The second V8 segment (V82) codes for a 8 peptide which is surfact -- expressed in the majority of TCR 81 ~ cells [ 11,19]. This peptide is recognized most likely by the anti-BB3 MoAb [ 11 ]. Based on the assignment of the epitopes recognized by these three MoAbs, namely anti-TcR 81 (C8), anti-STCSl (V8I }, and anti-BB3 (V82), double-color immunofluorescence analyses of cultured ct'!i lines and peripheral blood lymphocytes have indicated that the products encoded by either the VS1 or the V82 genes are surface expressed in more than 90c; ()t TcR ) , / 8 ~ peripheral lymphocytes [11,20,21].

To update our previous studies on the potential relation between human NK cell and early stages of T-lymphocyte maturation, we have assessed here th~ • genomic organization of the two predominant human V8 genes (i.e., VS1 a~ld V82) in a series of 19 cloned CD3 - N K cells.

MATERIALS A N D METHODS

Generation of Cloned Cell Lines

Cell lines were cloned by limiting dilution at 0.5 or 0.7 cells/well on a f~:eder layer of allogeneic irradiated Epstein-Barr v i rus- t ransformed B-cell line (LAZ 388) according to a technique previously described [3]. Clone 1 (TC48) wa~ obtained after bone marrow graft f rom a leukemic patient [22]. Clones 2, ~, l and 12 were derived from the peripheral blood cells of a 4-month-old child stimulated in secondary MLR. Clones, 4, 5, 8, 9, and 10 were derived from the sorted N K H 1 ÷ [2] peripheral blood cell fraction. Clone 6 is derived from normal fetal cord blood (23 weeks) as described previously [23]. Clone 7 ~JT3) was obtained from peripheral blood [24]. Clones 13 to 19 were derived from normal

V6 Gene Rearrangements in CD3- Cloned Lymphocytes 77

peripheral blood cells stimulated by phytohemagglutinin plus interleukin 2 and cloned after two cycles of depletion by immunorosetting with the TcR ~//3-spe- cific BMA031 MoAb [15].

Phenotypic Analysis of Cell Surface Antigens Phenotypic analysis was performed by indirect immunofluorescence with fluo- rescein-conjugated goat anti-mouse anti-serum as described previously [3]. Samples were analyzed on an Epics C (Coulter Electronics, Hialeah, FL) flow cytometer. Ten thousand cells were analyzed in each sample. MoAbs were always used at saturating concentrations ( 1 : 500 to 1 : 1000). Coulter clones CD2, CD3, CDd, CD8, and NKH1A were kindly provided by the Coultronics Company (Andilly, France). BMA031 MoAb [16], which recognized a monomorphic determinant on the TcR a//3 molecule, was kindly given by Dr. Kurrle (Behring Company, Marburg, Germany). Anti-NKH2 has been previously described [2]. The NKTa MoAb [25] was used as a negative control to determine background fluorescence.

Cytotoxici ty Assays

Cytotoxicity assays were performed according to a standard chromium release method described previously [3]. All experiments were done in triplicate using V-bottomed microtiter plats. Assays were generally performed for 3 hr at ~7°C using 5000 target cells per well.

Southern and Northern Blot Analysis

DNA was extracted from 3 × l ( f cloned cells, 12 to 14 days after plating. RNA was prepared from the cytoplasmic fraction of cells lysed in hypotonic buffer in the presence of Nonidet-P40. Southern and Northern blot analyses were carried out as previously described [ 11].

DNA Probes

~-'P-labeled DNA probes were prepared from agarose-purified DNA fragments by the hexamer priming method [26]. The V-specific TcR 6 probe cloned from the TirA + AB12 cell line (V62) has been described previously [11] anti corresponds to a 5' EcoRI-PvuI fragment derived from clone × 13, The V61-specific probe [8] is a EcoRI-ScaI fragment isolated from clone 0240/38.5 kindly given by Dr. Krangel (Dana Farber Institute, Boston, MA). The C6-spe- cific probe used in Northern blot analysis includes the 3' EcoRl fragment of clone ×13 [11].

RESULTS

We have performed the present study using a series of 19 well-characterized CD3 NK clones (Table 1) which represent, at least in part, the phenotypic heterogeneity [2,3] of human NK cells. These NK clones were selected on the differential expression of surface structures such as CD2, CDS, or NKH2, Note that 12 out of these 19 cells have been previously shown not to rearrange both the TCR/3 and TCR 3, loci [6].

In the Southern blots hybridized with the V62 probe, all the EcoRl-digested DNAs extracted from CD3 clones (six examples are given in Figure 1, panel A)

78 F. Triebet c{ al.

T A B L E 1 Surface pheno type and cytotoxicity against K562 of cloned cytotocic cell lines

Clones C D 3 ~ C D 4 T c R c~/~ N K H I CD2 C D 8 N K H 2 K52(,:

1 (TC48> ~ . . . . 2 (IlS5C) - - (+)' - 3 (IISXF) - + - 4 (T2IIBI) - + + 5 (T2IIC2) t 6 (F551IIE5) -- (+) ~- ÷ 7 (JT3) • + + 8 (T2IIA2) - ~ + ~- 9 (T2IH2) + , ~-

10 (T21FIO) -- ~ ~ l 1 (SIB5) - + + 12 (SIIH2) - + ~ + 13 (J2F4) - + ~ 14 (J1B2) ~ ' + 15 (N2EI(I) + 16 (NIA4) NI) ''~ ND ~ ~ ND 17 (N2G4) " ~ 18 (N1B2) ND ND + ~ ND 19 (N2B6) -- ND ND + + ND

• !

[ }

; z

N D N I )

N D N I )

N D N I )

Expression of antigens determined by indirect immunofluorescence reactivity with specific Mc)Abs

h Effector-to-target cell ratio I : 1 4-hr assays, ~ ~ ~- represents a percentage of specific cytotoxic~tv -50:~, ' > 2 0 ~ ; +, >109;.

Weak expression.

a Not done.

displayed a un ique germl ine band at 15 kb. As controls, two C D 3 : TcR /~1 clones were also tested with the same probe. The TcR 81 ) TiTA ~ AB 12 cell from which the V82 gene segment probe is derived has, in addi t ion to the ge rml ine signal, a 5.5-kb rearranged band that cor responds to the sequenced V - D - J ( j o i n ing)-C8 X13 c D N A [11]. The TcR 81 + TiTA-- c lone C1E10 has both chromosomes in ge rml ine configurat ion for the V82 gene (Figure 1, panel A, Lane 3). In contrast , when hybridized to the VS1 probe , the C1E10 clone D N A displayed the same V-D-J r ea r rangement described previously in the 1DP2 cclJ line [8], a 3.3-kb rearranged band (Figure 1, panel B, Lane 3) in addi t ion to the 3-kb ge rml ine signal. With the V81 probe no rearranged band was found m all the 19 C D 3 - clones examined (six C D 3 clones are shown in Figure l, panel B~ as well as in the T i r A ) clone AB 12.

To study the &chain t ranscript ional activity in the N K clones (three examples are shown in Figure 2, lanes 3, 4, and 5) we used a C8 probe. As a negative control , we used the K562 cell l ine (Figure 2, lane 2). In a separate expe r imen t we also used the V82 probe on the AB 12 R N A (Figure 2, lane 1) to define the respect ive posi t ions of previously sequenced ful l- length 2.2- and 1.3-kb V-D-J-C8 transcripts [ 1 t ]. These two transcripts cor respond to V-D-J-C tuRN As that use different sites of polyadenyla t ion in the 3' untranslated region as described previously [8,9]. In the C D 3 - cloned cells two signals, at 1.7 and 0.8 kb, were found (Figure 2, lanes 3, 4, and 5). These two signals cor respond to immatu re transcripts lacking V regions [8]. Thus , if addit ional , present ly u n k n o w n , V8 genes were rear ranged in some of these cells, they would not be transcribed.

V8 Gene Rearrangements in CD3- Cloned Lymphocytes 79

0 p " UJ M - . .

0 qc O 1 2 3 4 5 6

5.5--

A

F I G U R E 1 Southern blot analysis of TcR 8 gene rearrangement in 6 CD3 cloned lymphocytes with N K activity. The restriction enzyme used is EcoRl. Hybridizations were performed with the V82 probe (panel A) and with the V81 probe (panel B). Lane G is germline configuration. Control clones rearranged for either V82 or V81 included AB12, a TcR 81 + TiyA + clone, and C 1E 10, a TcR 81 ~ TiyA cloned cell line (see "Materials and Methods"). Restriction fragment sizes are given in kilobases (kb). Germline positions are 15 kb for V82 and 3 kb for V81.

D I S C U S S I O N

T h e genornic o rgan iza t ion o f the T c R / 3 and r genes in C D 5 - N K cells has been inves t iga ted p rev ious ly . T h e s e s tudies showed that these two genes are in ge rml ine conf igura t ion in bo th uncu l tu red whole N K cell f ract ions [4,5] and in a var ie ty o f c loned C D 3 - N K cell lip.es [6].

80 F. Triebel et al.

0 o I 2 3 4 § 6

B

F I G U R E 1 (Continued)

As shown by specific immunoprecipitations with anti-3' and anti-3 antisera, the' 3'/8 heterodimer is the first CD3-associated TcR to appear in murine ontogen3 (day 15), 2 days before the appearance of cell-surface TcR ~//3 heterodimers [27]. Early TCR 8 murine rearrangements have also been described using hybridomas derived from fetal thymocytes [28]. In human beings, fetal thymus DNA has been found to be rearranged at the eighteenth week of gestation m both the TCR 3" and 8 loci [29]. The analysis of the genomic organization of the locus is therefore relevant for the understanding of early events in T-celi differentiation.

To complete earlier observations made on the rearrangement status of TcR genes in cloned CD3 lymphocytes [6], we examined the TcR 8 gene organiza- tion in 19 CD3- cloned cell lines which are representative of the different phenotypes observed in unstimulated peripheral blood cells. For this purpose, we used as probes the two V8 segments, VS1 [8] and V82 [11], that code ik~r functional 8 chains surface-expressed on more than 90% of human peripheral blood lymphocytes [20].

V8 Gene Rearrangements in CD3 Cloned Lymphocytes 81

N N O

2 3

2 . 2 ""

1.7- 1.3-

0,0 ""

FIGURE 2 Northern blot analysis of CD3 cloned lymphocytes with a C8 probe. Total cytoplasmic RNAs from K562 and from three different CD3- clones were hybridized to a C8 probe. As a control for the full-length (V-D-J-C) transcript positions, we used the V82 probe (lane 1) that hybridized to the corresponding V8 transcripts in Abl2 cells. Transcript sizes are given in kilobases.

Our results, obtained with a large panel of clones from different origins, indicate that these two V8 genes are not rearranged in CD3 N K peripheral lymphocytes. However, it is likely that additional V8 genes will be described in the human. Our data suggest that such presently unknown V8 genes would not be transcribed in C D 3 - cells, which were shown to contain only 1.7- and 0.8-kb immature transcripts.

The present study was designed in an attempt to characterize further the status of TcR gene rearrangements in human N K cells. The data support the view that phenotypically distinct CD3 peripheral lymphocyte subsets displaying N K activity cannot be related to the T-cell lineage with the presently available V8 probes. Such results further strengthen the hypothesis that N K cells are likely to constitute a distinct lineage of cytotoxic effector cells even though some of these lymphocytes surface-express T-lymophocyte-related differentiation antigens such as CD2 or CD8 and transcribe the CD3~ gene [30]. Evidently, this question would have to be reevaluated if novel TcR loci are identified in humans, a

82 F. Triebel et ai

poss ib i l i ty that cannot be exc luded in l ight o f the r ecen t ident i f ica t ion in birds ot a po ten t ia l " th i rd" TcR chain (M. C o o p e r , unpub l i shed data).

A C K N O W L E D G M E N T S

This work was supported in part by the "Association pour la Recherche sur ic Can~et" grant no. 2 l 10.

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V8 Gene Rearrangements in CD3 Cloned Lymphocytes 83

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