Altered T cell receptor signaling and disrupted T cell development in mice lacking Itk

immunity, Vol. 3, 757-769, December, 1995, Copyright Q 1995 by Cell Press

Altered T Cell Receptor Signaling and Disrupted T Cell Development in Mice Lacking Itk

X. Charlene Liao’ and Dan R. Llttman’t l Department of Microbiology and Immunology tDepartment of Biochemistry and Biophysics University of California, San Francisco San Francisco, California 941438414 *Howard Hughes Medical Institute Molecular Pathogenesis Program The Skirball Institute of Biomolecular Medicine New York University Medical Center 540 First Avenue New York, New York 10016

Summary

Itk Is a T cell protein tyroslne klnase (PTK) that, along with Btk and Tee, belongs to a family of cytoplasmlc PTKs wlth N-terminal pleckstrln homology domains. Btk plays a critical role In B lymphocyte development. To determine whether Itk has an analogous role In T lymphocytes, we used gene targetlng to prepare mice lacking expression of Itk. Such animals had decreased numbers of mature thymocytes, an effect most clearly observed in mice expressing T cell receptor (TCR) transgenes. Mature T cells from Itk-deficient mice had reduced proliferative responses to allogeneic MHC stlmulation and to anti-TM cross-llnking, but responded normally to stimulation with phorbol ester plus ionomycin or with IL-2. These results provide genetic evidence that Itk Is involved in T cell development and also suggest that Itk has an Important role in proxl- mal events In TCR-medlated signaling pathways.

Introduction

Lymphocyte activation and development depend on receptor-initiated cascades of signal transduction events in- volving the action of several nonreceptor protein tyrosine kinases (PTKs). In mature T cells, stimulation of the T cell antigen receptor (TCR) by antigen and major histocompat- ibility complex (MHC) molecules or by antibody cross- linking induces PTK activation, which results in tyrosine phosphorylation of cellular proteins and subsequent activation of the phosphatidylinositol pathway, activation of Ras, and activation of several serine/threonine protein kinases and phosphatases (reviewed by Weiss and Littman, 1994). Similar intracellular signal transduction mechanisms are also likely to be involved in differentiation of immature thymocytes.

Nonreceptor PTKs involved in T cell activation and development include Lck and Fyn, members of the Src family of PTKs, and ZAP-70, which, along with Syk, forms a sepa- rate family of PTKs. Lck and Fyn are activated early after TCR cross-linking, and are thought to phosphorylate im- munoreceptor tyrosine-based activation motifs (ITAMs) within the TCR-associated CD3 and 6 polypeptides. ZAP-70 and Syk bind to the phosphorylated ITAMs by means of

SH2 interactions and are involved in subsequent activation of downstream components (reviewed by Weiss and Littman, 1994). Mice deficient in Lck have very few peripheral T cells or single-positive (SP) thymocytes and dramatically reduced numbers of doublepositive (DP) (CD4+CD8+) thymocytes (Molina et al., 1992). Expression of a mutant Ick transgene, which directs the synthesis of a dominant- negative form of the PTK, likewise results in early developmental arrest (Levin et al., 1993). Recent studies suggest that Lck is required for expansion of the DP population (Mombaerts et al., 1994). Mice with a targeted mutation in the gene encoding Fyn have a milder phenotype, with reduced thymocyte proliferation in response to TCR stimulation but no apparent defect in thymocyte development (Stein et al., 1992; Appleby et al., 1992). The discovery that mutations in human ZAP-70 result in a rare T cell immunodeficiency, which is manifested by the absence of mature CD8 cells and the failure of CD4 cells in the periphery to proliferate following TCR stimulation, indi- cates that ZAP-70 also has a critical role in thymocyte development (Arpaia et al., 1994; Chan et al., 1994; Elder et al., 1994). Studies of ZAP--/O-deficient mice generated by homologous recombination have confirmed and extended the role of ZAP-70 in thymocyte development. These mice have defective positive selection, with absence of mature CD4 and CD8 SP T cells, and defective negative selection, since antigen-specific ZAP-70+ thymocytes were not deleted in the presence of the peptide antigen (Negishi et al., 1995).

More recently, another nonreceptor PTKof yet unknown function was identified in T lymphocytes. This PTK was named Itk (interleukin-2 (IL-2)-inducible T cell kinase) be- cause its expression was induced by IL-2 in IL-2-deprived CTLL-2 cells (Siliciano et al., 1992). The same protein has also been designated Tsk (Heyeck and Berg, 1993) and Emt (Gibson et al., 1993; Yamada et al., 1993). Itkshares strong amino acid homology with two other mammalian proteins, Bruton’s tyrosine kinase (Btk) and Tee (Vetrie et al., 1993; Tsukada et al., 1993; Mano et al., 1993). These proteins define a new family of cytoplasmic protein tyrosine kinases, characterized by the presence of an N-terminal pleckstrin homology (PH) domain (Musacchio et al., 1993; Shaw, 1993) followed by the Src homology domains (SH3 and SH2) and a kinase domain. They lack the N-terminal myristylation signal and the C-terminal regula- tory tyrosine residue characteristic of the Src family of PTKs.

Btk is preferentially expressed in cells of the B lymphoid and myelomonocytic lineages. Mutations in Bfkare associated with X-linked agammaglobulinemia (XLA) in human (Vetrie et al., 1993; Tsukada et al., 1993) and X-linked immunodeficiency(xid) in mice (Thomas et al., 1993; Raw- lings et al., 1993). The defects in XLA and xid are both intrinsic to the B lineage, but the phenotypes differ significantly. Whereas XLA patients have few circulating mature B cells, xid mice, which are homozygous for a mutation in the Btk PH domain, have only a moderate defect in B

cell maturation, but have defects in B cell activation and in the production of some immunoglobulin isotypes (reviewed by Tsukadaet al., 1994). Interestingly, a null mutation in Btk results in a phenotype similar to xid (Khan et al., 1995). Stimulation of the antigen receptor on B cells and of the immunoglobulin E (IgE) receptor on mast cells results in tyrosine phosphotylation and increased PTK ac- tivityof Btk(Saouaf et al., 1994; Aoki et al., 1994; de Weers et al.; 1994; Kawakami et al., 1994).

Itk is expressed preferentially in cells of the T lineage (Siliciano et al., 1992; Heyeck and Berg, 1993; Gibson et al., 1993). Its tissue distribution is restricted to thymus, lymph node, and spleen. In thymus, Itk mRNA has been detected at day 14 of fetal development, prior to the appearance of DP thymocytes (Heyeck and Berg, 1993). Itk mRNA has also been detected in human natural killer (NK) cell lines (Gibson et at., 1993; Tanaka et al., 1993) but not in murine NK cells (Desiderio and Siliciano, 1994). Stimulation of T lymphocytes with anti-TCR or anti-CD28 antibodies results in phosphorylation and activation of Itk (August et al., 1994).

The strong homology between Btk and Itk and their apparently similar modes of activation following antigen receptor engagement in B and T cells, respectively, have suggested that these PTKs have similar functions in the two lymphocyte lineages. To identify the functions of Itk and, in particular, to ask whether Itk plays an analogous role in T cells to that of Btk in the development of B cells, we generated mice with a null mutation in the itk gene. Mutant mice exhibited decreased numbers of mature T lymphocytes and, more strikingly, they had a nearly complete block in development of thymocytes bearing,defined transgenic TCFls. Mature T cells in mutant mice displayed impaired TCR-mediated proliferation. Our studies thus indicate that Itk has an important role in thymocyte maturation, most likely through its involvement downstream of TCR-initiated signals.

Results

Generation of Itk-Deficient Mice Several overlapping genomic clones oi the if/r gene were obtained from a LDASH II phage library derived from the 129/Sv mice. Three large clones defined a contig of 20.5 kb encompassing the putative exons 2, 3, 4, and 5. The putative exon 1 containing the translation initiation codon was not included in these genomic clones. We identified two splice acceptor sites preceding exon 3 (Figure 1A). The alternative use of the these splice sites 18 bp apart may explain the discrepancy of the published cDNA se- quences of itk (Siliciano et al., 1992; Heyeck and Berg, 1993; Yamada et al., 1993). The organization of the 5’end of the itk gene is very similar to that of the mouse and human Bt/rgenes(Rawlings et al., 1993; Ohta et al., 1994; Hagemann et al., 1994; Sideraset al., 1994; Rohrer et al., 1994). These two genes may be derived from duplication of a common ancestor.

The scheme used to generate a null allele of the itkgene is illustrated in Figure 18. The putative exon 4 and portion of exon 3, as well as adjacent introns of itk were deleted

and replaced with pgk(G)neopA. The deleted itk coding sequence includes a region that is highly conserved among the ltk/Btk/Tec family members. This region contains a tryptophan residue that is the only absolutely conserved amino acid among all known PH domains (Musac- chio et al., 1993). The poly(A) addition signal present in the neo cassette was expected to terminate pre-mRNAs from both the pgk anditk promoters, so that no Itk protein would be produced. To ensure further a null allele, a stop codon was engineered at a position in the itk gene corre- sponding to amino acid 78, upstream of the deleted exons (Figures 1A and 1 B). The itk mutation was introduced into the mouse genome by standard techniques using the Jl embryonic stem (ES) cell line, which was derived from the mouse strain 129/Sv (Li et al., 1992). Homologous recombination at theitklocus wasdetected by polymerase chain reaction (PCR) and confirmed by Southern blot analyses using the 5’ and 3’ probes (Figures 1 B and 1 C; data not shown), both of which lie external to the homologous regions in the gene-targeting construct. The targeted ES cells were used for blastocyst injection. The chimeric animals generated were subsequently back-crossed to C57BU 8J mice and agouti progeny bearing the targeted itk allele were interbred for homozygosity of the targeted allele.

Thymocytes from the wild-type mice were lysed and im- munoblotted with rabbit antisera specific for Itk. Murine Itk exhibits an apparent molecular mass of 72 kDa (Figure lD, lane 5; see also Siliciano et al., 1992). No Itk protein product was detected in thymocytes from mice homozygous for the mutated itk gene (Figure 1 D, lane 7). More- over, thymocytes from heterozygous mice exhibited levels of Itk approximately half relative to normal mice (Figure lD, lane 8). The location of Itk on the SDS-PAGE gels was confirmed by analyzing COS cells transfected with full-length murine itk cDNA (Figure 1 D, compare lanes 3 and 4 with lanes 1 and 2). Two different rabbit polyclonal antisera, ItkN4 and ltkCL45, were used for Western blot detection of Itk and gave similar results (data not shown for ltkCL45).

To investigate whether truncated forms of the Itk protein bearing epitopes recognizable by one but not the other Itkantiserum were produced in it/c- mice, Itk was immunoprecipitated from thymocyte lysates with ItkN4 and was detected by immunoblotting with ItkN4 or ltkCL45. Under such conditions, thymocytes from the wild-type mice gave rise to a single protein band with a molecular mass of 72 kDa (Figure 1 E; data not shown). Reciprocally, using ltkCL45 for immunoprecipitation followed by blotting with ltkCL45 or ItkN4 also gave the same result (data not shown). Mice homozygous for the targeted allele of itk had no detectable Itk protein of any size by immunoprecipitation and immunoblotting (Figure 1 E, lanes 5 and 8). Thus, the mutation of itk represents a null allele.

T Cell Development in the Absence of Itk Itk-deficient mice were viable and fertile, and had no evidence of gross developmental defects. The total number of thymocytes in mutant mice was similar to that in heterozygous or wild-type littermates (Table 1). Moreover, the

;gCgell Phenotype in Itk-Deficient Mice

B %*

2 3 4 5

73 th 12.9 kb 4.9 kb 6.9 kb I

8 4.9kb v% k

size

H wr

H rulant

C D Pnim ltkN4

E I- +,+ +,+ +,+ *I+ A. J- +,+ +,+ +,- +,- 4 J-

cos

klrdKD- -ftk

e* KD- --ttk 1234567

,23456 44KO-

1 2 345s

Figure 1. Targeted Disruption of the itk Gene (A) Comparison of intron placement in the murine Ilk and Bt/r genes. Exon 1 of the mouse i&gene is shown here to contain the translation initiation codon. Mouse and human Btk have the same organization for intron/exon bound- aries and the initiation codon was found to be in exon 2 of Btk (Rawlings et al., lsg3; Ohta et al., 1994; Hagemann et al., 1994; Sideras etaf.,1994;Rohreretal.,1994).TheAOandA1 denote the type of intron boundary (between codons, or after the first nucleotide of the codon, as indicated with a vertical bar). The conserved amino acids between Itk and Btk are shown in bold and gaps in the amino acid se- quences are represented by dashes. The 8 ex- tra amino acids in the longer isoform of the murine ihk cDNA are underfined. The position of the stop codon in the MI gene-targeting construct is shown with an asterisk above Tyr78. (B) Partial restriction map of the Mk gene. Both the wild-type allele and the targeted allele of if/r are shown, together with a diagnostic scheme for their differentiation. The horizontal lines refer to introns, and boxes refer to exons. The fragments involved in homofogous recombination are represented with thick lines. Re striction sites unique to the targsted allele of it& are circled. The arrowheads indicate prlm- ers for PCR screening of homologous recombination in the ES cells. The external probes (5’ probe and 3’ probe) used in Southern blot analysis to confirm homofogous recombination and

to screen for mice with the itk gene disruption are indicated with hatched boxes. Asterisk, an in-frame stop codon created in the targeted allele of iti. B, BamHI; H, Hindlll; X. Xbal. (C) Analysis of tail DNA samples from mice derived from an ES clone carrying the disrupted allele of the if/r gene (with the designed stop codon at exon 2) using the 5’ probe. After Hindlll digestion, the wild-type allele of itk is represented by the top band (25.1 kb) and the mutant allele by the bottom band (17.8 kb). (D) Western blot analysis of Itk in COS cell or thymocyte lysates. COS cells were transiently transfected with expression vectors without (lanes 1 and 2) or with the full-length cDNA of murine it/c (lanes 3 and 4). Thymocytes were from mice with the following genotypes at the if/r locus: +I+. lane 5; +I-, lane 8; and -/-. lane 7. Antiserum ItkN4 (against amino acids 1-175 of ltk) was used at 15000 in TBST with 5% nonfat dry milk. Similar results were obtained with antiserum ltkCL45 (against amino acids 175-819 of Itk). The position of Itk is indicated (lower band in the doublet). (E) lmmunoprecipitation and Western blot analysis of Itk in thymocyte lysates. Thymocyte lysates from if/V mice (lanes 1, 2, 3, and 4) were immunoprecipitated with ItkN4 (Lanes 2, 3, and 4) or preimmune serum from the same rabbit (lane 1). Thymocyte lysates from it/r- mice (lanes 5 and 8) were immunoprecipitated with ItkN4. The immune complex was resolved by 7.5% SDS-PAGE with reducing agent and blotted for Itk with ltkN4. The position of Itk is indicated. Rabbit immunoglobulins account for the strong background signal.

percentage of double-negative (DN) and DP thymocytes heterozygous or wild-type littermates (Table 1; Figure 2A). was comparable in all the mice (Table 1). However, the This suggested that development of CD4 SP thymocytes ratio of CD4 SP versus CD8 SP thymocytes in I&-‘- mice was more sensitive to the lack of Itk than that of CD8 SP was reduced 2- to 3-fold when compared with the ratio in thymocytes. Although the overall cell numbers in lymph

Table 1. T Cell Populations in the Thymus and Lymph Nodes of Itk-Deficient Mice’

Total Cells Percentage of Total T cells* Percentage of Total Cells (%)

CD4lCDS Genotype Source x 10-r T Cells (%) x 10-7 CD4-CDS- CD4+CDS+ CD4+CDS- CDCCDS’ Ratio

itk+‘+ (n = 2)b Thymus 25.5 A 10.8 2.3 f 0.1 80.5 f 3.0 12.0 f 1.7 5.2 f 1.2 2.3 f 0.2 itk”- (n = 5)b Thymus 20.2 f 7.8 2.8 f 0.8 82.8 f 2.8 10.4 f 1.4 4.2 f 1.1 2.8 * 0.4 it/r- (n = 8)b Thymus 18.8 f 8.9 3.2 f 0.7 79.8 zt 3.2 7.9 f 0.8 9.1 f 3.4 0.9 * 0.3 it/r+‘+ (n = 8) Lymph nodesd 5.2 f 2.1 75.5 f 8.3 3.7 f 1.5 4.4 + 2.9 0.5 f 0.2 50.9 f 4.7 19.7 f 7.8 3.3 f 2.4 it/r+- (n = 14)c Lymph node@ 5.1 zt 2.0 70.8 f 8.4 3.8 f 1.2 3.5 Et 1.3 0.4 f 0.2 45.1 f 8.4 21.7 f 5.8 2.4 f 1.5 irlr’- (n = 18)c Lymph nodes” 4.7 f 4.2 47.1 f 12.4 1.9 f 1.7 8.4 f 2.3 0.2 f 0.1 21.2 f 8.2 19.4 f 7.9 1.2 f 0.4

n Data are expressed as mean f standard deviation. b Mice, 5-8 weeks old; numbers of animals studied are shown in parentheses. c Mice, 3-t 3 weeks old; numbers of animals studied are shown in parentheses. d Cervical; brachial; axillary; superficial inguinaf, and mesenteric. e The number of T cells in the lymph nodes was calculated based on the percentages of cells staining positive with antiCDh.

Immunity 760

nodes and spleens remained unchanged, the number of T cells was reduced by 15 to P-fold in ltkdeficient mice, compared with that in heterozygous or wild-type littermates (Table 1). The number of B cells was increased correspondingly in Itkdeficient mice, asjudged by staining with the B cell markers 8220 (Figure 28) and IgM (data not shown). Consistent with the reduced ratio of mature CD4+ versus CD8+ thymocytes in the homozygous mutant mice, the number and proportion of CD4+ T cells in the periphery suffered a moresevere reduction compared with that of the CD8+ T cells (Table 1).

Responses of T Cells from ltk-Deficient Mice to Allogeneic MHC Challenge To determine whether T ceils that appear in the periphery of Itk-deficient mice are functional, we assayed their proliferation in response to allogeneic stimulation. To adjust for the difference in the number of mature T ceils from ifk’- and control if/P’- littermates, lymph node cells depleted of B cells and CD8+ T cells were used as responder ceils in these assays. They contained about 90% CD4+ T ceils with similar surface TCR level, and were of the H-2b haplotype. When tested in mixed lymphocyte reaction

Figure 2. Thymue and Lymph Node Profiles of Itk-Deficient Mice Flow cytometric analyses of thymcqtes (A) and lymph node cells (2) from 5weekold wild type (HP+), heterozygous (ilk”), and homozygous (rtrcl-) llttetmates stalned with an&CM and anti-CD& or anti-CDS and anti-2220. Per- cent of total cells in each quadrant is indicated.

(MLR) with irradiated BALB/c splenocytes (H-P), the CD4’ T cells from the homozygous mutant mice showed significantly reduced proliferation compared with heterozygous littermates (Figure 3). Separately purified CD4+ and CD8+ cells from Itkdeficient mice and control littermates were also challenged with irradiated splenocytes from the mouse strains B8.H-2bm12 (class II mutant) and B8.H-2bm1 (class I mutant), respectively. Reduced proliferation was observed with both CD4+ and CD8+ cells from Itkdeficient mice compared with the control mice (data not shown). Taken together, these data indicate that T cells in the pe- ripheryof itkdeficient mice are compromised in their proliferative responses elicited by ailoantigenic stimulation.

TCR-Mediated Proliferative Responses in ltk-Deficient Mice To characterize further the cell proliferation defect in Itk- deficient animals, CD4+ T cells were stimulated with con- canavalin A (ConA) or anti-CD3 antibodies in the presence of irradiated syngeneic splenocytes. Significant reduction in proliferative responses to both stimuli was observed with cells from the mutant mice compared with those from control littermates (Figures 4A and 48). However, cells

T Cell Phenotype in Itk-Deficient Mice 761

2 -- + I&-, with BALE/c

ii

--& Id-, with BALBk + ltk +I-, with C57BlJB

.E -- --D-, /fk-‘-, wtthC57B116

iii ‘0 2 0 0 --

E 1 //I

jyyd 0 1 2 3 4

# of Responders x 1W5 Figure 3. Proliferative Response towards Allogeneic MHC

CD4+ lymph node cells from mice of the indicated genotypes (W-, solid lines; and iti-‘-, broken lines) were stimulated in vitro with irradiated splenocytes from C57BU6 mice (squares) or from BALBlc mice (triangles). Cell proliferation was measured by PHjthymidine incorpora- tion. Standard deviations for triplicate samples are shown as error bars along they axis, some of which may be not apparent due to their small values.

from itk+ mice and itk+‘- mice responded similarly to phorbol myristate acetate (PMA) and ionomycin (Figure 48). These data suggest that T cells lacking Itk are compromised in a relatively early phase of the TCR-mediated signaling pathway.

Based on induction of itk mRNA in CTLL-2 cells with IL-2, it was suggested that Itk may mediate IL-2R signaling (Siliciano et al., 1992). To look at T cell reactivity to IL-2, exogenous IL-2 was added to the culture together with a submitogenic dose of ConA. Under such conditions, both irk’- and iW- cells responded to addition of exogenous IL-2 with enhanced proliferation (Figure 4C). These data indicate that Itk is not essential for IL-2R signaling and suggest that reduced production of IL-2 by irk’- T cells may be a primary reason for their reduced proliferation.

Effect of Loss of Itk on T Cell Development in TCR Trenegenic Mice Mice expressing specific transgenic TCRs have provided valuable insight into the mechanisms of positive and negative selection and the role of individual gene products in these processes. To determine whether Itk has a role in the selection of T cells having a single specificity, we introduced transgenic TCRs specific for either MHC class II or class I into the Itk-deficient background. Positive Selection of T Cells Expressing Class /l-Restricted 7CR Transgenes AND TCR transgenic mice express a TCR that is specific for pigeon cytochrome C (PCC) bound to the MHC class II molecule I-Ek (Kaye et al., 1989). Positive selection of AND T cells in the H-2k or H-2b background results in the appearance of mature T cells that are virtually all CD4 SP and express high levels of both transgenic TCR a and 8

A

1 0.5 0.25 0.125 ConA @g/ml)

B

iluLL- ‘Ooom

0 0 1.5 0.3 0.167 0.050

I anti-CD3 @g/ml) PMA + lonomycin

Figure 4. Cell Proliferation Induced by TCR Stimulation

Purified CD4’ lymph node cells were stimulated with ConA (A) or purified anti-CD3 (B) at the indicated concentrations, or with PMA (1 ngl ml) and ionomycin (500 nglml) (B). Alternatively, they were stimulated with 0.125 pg/ml ConA plus indicated concentrations of human recombinant IL-2(C). Cell proliferation was measured by PH]thymidine incor- poration. Standard deviations for triplicate samples are shown as error bars along the y axis.

polypeptides (Kaye et al., 1992). In the absence of Itk, there was a dramatic reduction in the proportion and numbers of CD4+CD8- thymocytes in the AND TCR transgenic mice (Figure 5A; Table 2). Consistent with the phenotype observed in the thymus, in the periphery of if/c’- AND TCR mice there was a profound reduction in the number of CD4+ T cells (Table 2; Figure 58). The few CD4+ cells that developed in the mutant mice had a significant reduction in expression of the TCR a chain (40%-50% versus more than 90% in heterozygous littermates), suggesting that these cells were selected due to expression of endogenous TCR a chains (Figure 58). Concomitant with the reduction in CD4’ T cells; there was a 5- to 7-fold increase in the number of CD4CD8- T cells in it/r-- AND TCR mice

Immunity 762

A AND Tg, it& +I-

CDS - I-Restricted TCR Transgenes

CM+CDs- l’hymecytes

83.1%

To ask whether Itk also affects development of CD8 lineage cells, we studied positive selection of T cells expressing class l-specific TCR transgenes in the Itkdeficient background. Thymocyte development is skewed towards the CD8 lineage in female H-2b mice expressing a transgenic TCR specific for the male-specific H-Y antigen and the class I molecule Db (von Boehmer, 1990). This skewing is not as dramatic as it is for the CD4 lineage in the AND mice; nevertheless, selection of thymocytes bearing the clonotypic TCR can be followed using the T3.70 monoclonal antibody (Teh et al., 1988,1989). In the absence of Itk, there was a decrease in the efficiency of selection of CD4-CD8+T3.70hi thymocytes (Figure 6A). This effect was particularly evident in the periphery of these mice, where the number of CD4CD8+ T cells bearing the clonotypic TCR was greatly reduced despite little difference in the total number of CD4CD8+ cells (Figure 6B; Table 2). Mature CD4+ T cells appear in the periphery of H-Y transgenic mice due to rearrangement at the endogenous TCR a locus (von Boehmer, 1990). In female if/P H-Y mice, selection of both the CD4 and CD8 T cell lineages thus favored cells that utilized endogenous TCR a chains.

B AND Tg, It& +I-

Lymph Nodo

AND Tg, Itk -‘- compared with the control AND TCR mice (Table 2). These CD4CD8- T cells also expressed the transgenic TCR a and 5 polypeptides (Figure 58; data not shown). These data suggest that Itk is involved in development of CD4’ T cells. Its absence dramatically changed the developmental outcomes of T cells bearing a defined TCR, resulting in appearance of only a few CD4+ cells, most of which had lost expression of the clonotypic receptor, or of cells that had lost coreceptor expression but retained expression of the transgenic TCR.

Positive Selection of T Cells Expressing Class

“1 ( I

AND Tg, It& -‘-

Figure 5. Absence of Itk Interferes with Development of CD4 Lineage Cells in AND Transgenic.Mice

(A) Thymocytes from AND transgenic Mr+- and AND transgenic if/r- mice (both H-p) were stained with antiGD4, antiCD6, and anti-Val 1, which recognizes the transgenic TCR a chain. Expression of CD4 and CD8 is shown as dot plots (top). Percent Of total cells in each quadrant is indicated. CD4+CD8- cells in the upper,left quadrant were electronically gated to show expression of the transgenic TCR a chain (Vail histogram, bottom). Proportion of Val I+ cells (as marked by Ml) in the gated population is indicated.

The number of CD4’ T cells in the periphery of female irk’- H-Y TCR mice was reduced compared with heterozygous (ifP) H-Y TCR littermates (Figure 6B; Table 2), indicating that development of CD4+ T cells in general contin- ued to be affected by the absence of Itk. This is consistent with phenotypes found in nontransgenic it/c- as well as it/c- AND TCR mice. In contrast with it/r-‘- AND TCR mice, the number of DN T cells in the periphery of female it/r’- H-Y TCR mice was not significantly different from that of if/r+‘- H-Y TCR mice or from nontransgenic mice (Table 2; data not shown). Negative Selection of Thymocytes Expressing the H-Y-Specific TCR Transgenes The H-Y transgenic mice also provide a system to ask whether Itk is involved in negative selection of thymocytes. In male transgenic mice, thymocytes expressing the H-Y- specific transgenic TCR interact with the male-specific an-

(B) Lymph node cells of the AND transgenic it/r+- and AND transgenic if/r+ mice (both H-29 were stained with antiCD4, anti-CD& and anti- Val 1 as in (A). Expression of CD4 and CD8 (top), as well as expression of CD4 and Val 1 (bottom), is shown as dot plots. Percent of total cells in each quadrant is indicated. The lower right quadrant of the bottom panel contains cells that were CD4-CD6-Vail’.

;6pll Phenotype in Itk-Deficient Mice

Table 2. T Cell Populations in the Thymus and Lymph Nodes of the AND TCR and H-Y TCR Transgenic Mice’

Total T cells* CDCCDB- CD4’CDB’ CD4+CD0- CDKCD8’ Genotype Source x 10-7 x lo+ x10-’ x10* x10*

AND transgenic, it/r+- (n = 3)b Thymus 20.7 f 3.8 14.2 f 1.3 10.4 f 2.7 79.1 f 15.7 9.2 f 5.4 AND tranegenic, if&- (n = 8y Thymus 18.2 f 9.6 23.4 f 12.0 14.6 k 8.0 9.3 f 7.1 3.7 f 3.0 AND transgenic, if/f- (n = 3)” Lymph nodesd 3.7 f 2.3 3.5 f 3.6 - 31.0 f 18.5 1.3 f 0.5 AND transgenic, if/r- (n = 8)b Lymph nodesd 3.4 f 1.5 24.2 f 12.8 - 5.9 f 2.7 3.7 f 2.2 H-Y transgenic, if/@ (F, n = 4) Thymus 20.2 f 7.2 11.5 f 7.6 13.4 f 6.7 21.6 f 11.4 34.6 f 13.0 H-Y transgenic, if/r- (F, n = 4) Thymus 15.5 f 6.7 16.1 f 12.0 11.2 f 6.0 11.4 f 6.5 15.8 f 10.5 H-Y transgenic, it/@ (F, n = 4) Lymph node& 3.6 f 0.9 1.0 f 1.2 - 23.9 f 7.2 10.5 f 1.0 H-Y transgenic, it/r- (F, n = 4) Lymph nod& 1.9 f 0.6 2.7ztl.5 - 9.4 f 2.9 6.7 f 1.6 H-Y transgenic, if/r+- (M, n = 5)” Thymus 2.2 f 0.9 15.9 f 7.3 0.10 f 0.07 3.7 zt 1.6 1.7 f 0.9 H-Y transgenic, it@- (M, n = 4)’ Thymus 3.0 f 1.4 13.7 f 7.7 0.68 f 0.36 4.9 f 0.8 4.9 f 3.7 H-Y transgenic, iW- (M, n = 5) Lymph node@ 1.5 f 0.3 9.1 f 3.5 - 0.8 f 0.3 5.1 f 1.6 H-Y transgenic, irks- (M, n = 4) Lymph nodesd 1.2 f 0.3 3.9*1.3 - 1.2 f 0.5 6.6 f 2.6

’ Data are expressed as mean f standard deviation. Dashes indicate negligible numbers with no significant difference among the animals. b Mice, 8-10 weeks old; numbers of animals studied are shown in parentheses. c Mice, 8-10 weeks old; the sex and numbers of animals studied are shown in parentheses. F, female; M, male. @ Cervical, brachial, axillary, superficial inguinal, and meeenteric. a The number of T cells in the lymph nodes was calculated based on the percentages of cells staining positive with anti-CD&.

tigen presented by H-2Db, resulting in early negativeselec- tion. Deletion in the TCR transgenic mice is accompanied by a large decrease in the size of the thymus, primarily owing to a reduction in the total number of CD4+CD8+ thymocytes (von Boehmer, 1990). Although there was a consistent increase in the fraction of CD4+CD8+ thymocytes in male H-Y transgenic it/r-‘- mice compared with H-Y transgenic it/f+- mice (Figure 7; Table 2) the total number of thymocytes was unaffected (Table 2). In the periphery of male H-Y transgenic mice, self-reactive CD8+ T cells escape clonal deletion by down-modulating CD8 expression (Teh et al., 1989). This population of lymph node T cells was also present in mice lacking Itk, and no CD4-CD8hiT3.70+ cells were observed (data not shown). These results suggest that Itk does not play a major role in negative selection of self-reactive thymocytes.

Discussion

Signaling pathways following activation of thymocytes and T lymphocytes remain incompletely understood. Although there is general consensus that early signaling involves the activity of Src family PTKs and of ZAP-70, it is not yet clear how downstream effector pathways are accessed and modulated. Moreover, it remains unclear what types of signaling difference are involved when activation of thymocytes results in different developmental outcomes, such as positive selection versus induction of programmed cell death (reviewed by Rothenberg, 1994). Lck is required for effective TCR-mediated T cell activation (Straus and Weiss, 1992; Karnitz et al., 1992) and for expansion of CD4+CD8+ thymocytes in thymocyte development (Molina et al., 1992; Mombaerts et al., 1994). ZAP-70 is required later, for the progression of DP thymocytes to mature T cells (Arpaia et al., 1994; Negishi et al., 1995). In this re- port, we have explored the role of ltk, a member of a third class of cytoplasmic PTKs that is expressed in T lymphocytes, in T cell activation and development. We have shown that mice lacking Itk have a moderate reduction in the number of mature T cells, particularly CD4 SP cells.

This effect was dramatically magnified when we analyzed development of T cells with a defined transgenwncoded TCR. Furthermore, mature T cells in the ltk-deficient mice were impaired in their proliferative responses to allogeneic MHC, anti-CD3 antibodies, and ConA. This study hence identifies Itk as another PTK with an important role in T lymphocyte signal transduction.

A Partial Block of T Cell Development in Itk-Deficient Mice Itk-deficient mice had reduced numbers of mature thymocytes and lymph node T cells, as well as a reduced ratio of CD4 to CD8 SP cells in thymus and, to a lesser degree, in the periphery. This could reflect either reduced production or reduced survival of mature T cells in the absence of Itk. Although a more complete developmental block might have been expected based on the effects of Btk mutations in human XLA patients, a partial block in T cell development in Itk-deficient mice more closely resembles the mild phenotypes of Btk mutations in mice. xid mice with a point mutation in the PH domain of Btk, as well as mice with a targeted insertional mutation in the Bfk gene (Khan et al., 1995) have only a moderate reduction in numbers of mature B cells, but these cells are defective in their proliferative responses to surface immunoglobulin cross-linking (Mond et al., 1983; Khan et al., 1995). The partial block in T and B cell development in mice that have defects in Itk and Btk, respectively, may be due to overlapping functions of related PTKs expressed in these cells. For example, Tee, the other member of this family of PTKs, was shown to be expressed in thymus(Mano et al., 1993). Since these and potentially other family members may compensate for the absence of Itk, it will be important to study mice defective for two or more members of this family of kinases.

Development of T Cells with Tranegenic TCRs in the Absence of Itk The absence of Itk had a much more profound effect on the development of T cells bearing a defined transgene-

Immunity 764

A H-Y Tg, itk +I-

H-Y Tg, Itk +‘- H-Y Tg, Ifk -I-

H-Y Tg, itk -‘-

- 20.81 21.11 16.31 10.9

85.5% 65.6%

i T3.70 -

Figure 7. Clonal Deletion of the H-Y-Specific TCR in the Absence of Itk

Thymocytes from male H-Y transgenic it/r+ or itk’- mice (both H-2”) were stained with antiCD4, anti-CD& and biotinconjugated T3.70. specific for the transgenic TCR a chain. Expression of CD4 and CD3 is shown as dot plots (top). Percent of total cells in each quadrant is indicated. Expression of the self-reactive transgenic TCR on total thymccytes is shown at the bottom.

B H-Y Tg, itk +‘- H-Y Tg, Itk -I-

Lymph Node

encoded TCR than on the broad repertoire of T cells. In mice expressing the class II-restricted AND receptor, loss of Itk resulted in a reduction of more than 80% of mature CD4+ T cells (Table 2; Figure 58). The loss may be due to an early block in the transition from DP to SP thymocytes (Figure 5A). Loss of Itk may thus result in reduced signal transduction upon TCR engagement, with decreased proliferative capacity as well as reduced positive selection of CD4+ thymocytes.

Alternative explanations are suggested by the observations, in the periphery of it/r’- AND TCR mice, that most of the remaining CD4CDS cells have lost expression of transgenic TCR and that there is a significant increase in the number of CD4CD8- cells expressing the transgenic TCR (Figure 58). CD4CD8- T cells expressing the transgenic TCR may represent a lineage distinct from con- ventional TCRa5 T cells. Similar DN populations in other TCR transgenic mice appear to be independent of positive

CM‘CDa+ Lymph Ncde Cells

recognizes the transgenic TCR a chain. Expression of CD4 and CD3 is shown as dot plots (top). Percent of total cells in each quadrant is indicated. In each case, CD4CD8’ cells in the lower right quadrant were electronically gated to show expression of the transgenic TCR as stained with T3.70 (bottom). Proportion of T3.70’ cells (as marked by Ml) in the gated population is indicated.

Figure 6. Positive Selection of CD8 Lineage Cells in the Absence of Itk

Thymocytes (A) and lymph node cells (6) from the female H-Y transgenic it/r+- and H-Y transgenic itk-‘- mice (both H-2”“) were stained with antiCD4, anti-CD& and biotin-conjugated T3.70, which


or negative selection (Russell et al., 1989; Robey et al., 1992a). The lack of Itk may then serve to reveal an otherwise underrepresented lineage in T cell development.

Another alternative explanation derives from comparison with observations of DN T cells in transgenic mice that express a self-reactive transgenic TCR (Schonrich et al., 1991) or that overexpress a coreceptor in combination with acognate TCR (Lee et al., 1992; Robeyet al., 1992b; Davis and Littman, 1995). In the latter cases, increased avidity due to the contribution of coreceptor to TCR interactions with MHC can result in negative selection; loss of either the coreceptor or of the TCR can apparently allow for positive selection of the thymocytes. Our observation of CD4-CD8- T cells expressing transgenic TCR in the periphery of itk’- AND TCR mice therefore raises the possibility that the decrease in CD4 lineage cells in it/@- AND TCR mice is due to increased TCR signaling during development in these animals. Itk may thus normally down- modulate TCR signals and its absence may allow enhanced negative selection. However, there was no obvious enhancement of CD3-mediated signal transduction in thymocytes from Itk-deficient mice, as measured by proliferation and accumulation of intracellularfreecalcium (data not shown). Moreover, there was no apparent difference in the proportion of DN to DP thymocytes between control and Itk-deficient animals (Table l), and lack of Itk did not result in increased negative selection in male H-Y transgenic mice, which takes place early in thymocyte development. Therefore, for this scenario to be correct, enhancement of negative selection would have to occur in a narrow window late in thymic development.

In mice transgenic for the MHC class l-specific H-Y TCR, there is positive selection of clonotype-positive CD4-CD8+ thymocytes in female mice of the appropriate MHC background. However, in contrast with the AND mice, a large proportion of developing thymocytes in the female H-Y mice are also selected on the basis of expression of endogenous TCR a chains. In the absence of Itk, there was dramatic loss of the CD8 lineage thymocytes and mature T cells expressing the T3.70+ clonotypic TCR, but apparently normal development of CD8 lineage cells that utilize endogenous TCR a chains (Figure 6). This result may be due to a defect in positive selection of CellS

expressing the transgenic TCR in the absence of Itk. Alter- natively, based on the results in the AND transgenic mice, this could be due to enhanced negative selection, oc- curring late in thymocyte development, of cells expressing the clonotypic TCR. In the AND mice, this would be manifested by a dramatic decrease in the CD4 SP population and an increase in clonotype-positive DN T cells; in the H-Y mice, this could result in appearance of only clonotype-negative cells.

Role of Itk in T Cell Signal Transduction T cells from Itk-deficient mice had reduced proliferative responses to allogeneic MHC stimulation and to TCR cross-linking. Since these cells proliferated as well as cells from control mice in response to PMA and ionomycin, which bypass stimulation of cell surface components, Itk

appears to be involved in membrane-proximal events in T cell activation, downstream of a TCR-initiated signal. Earlier studies have suggested that Itk may be involved in other signaling pathways. There was an increase in itk mRNA upon treatment of CTLL-2 cells with IL-2 (Siliciano et al., 1992), suggesting a possible role for Itk in IL-2 recep tor signaling or IL-2-dependent effector functions. We found that the decreased proliferative response of Itk- deficient T cells to ConA was completely rescued by addition of exogenous 11-2, indicating that Itk is not required for signaling through the cytokine receptor. Itk has also been shown to be coprecipitated with CD28 and to become phosphorylated following CD28 cross-linking (August et al., 1994); it has thus been postulated that Itk has a key role in CDS&mediated costimulation. In preliminary studies, we have found that the CD28 signaling pathway is not impaired in T cells from the Itkdeficient mice (X. C. L., S. Fournier, J. Allison, and D. R. L., unpublished data). In addition, ltk-deficient mice have a phenotype distinct from that of CD28deficient mice. For example, unlike Itk- deficient animals, mice lacking CD28 have normal T cell development, with normal numbers of mature T cells in the periphery (Shahinian et al., 1993). In addition, CD2& deficient mice have abnormal immunoglobulin isotypes, which were not observed in Itkdeficient mice (unpublished data).

Our results are consistent with a role for Itk in efficient TCR-mediated signaling that results in proliferative responses. However, our analysis of mature T lymphocytes employed cells that had undergone thymic selection in the absence of Itk. We must, therefore, consider the possibility that reduced responses of Itk-deficient T cells may reflect a compensation that permits selection of these cells. If Itk normally delivers a down-modulatory signal during T cell development, as was suggested above, positive selection of Itk-null thymocytes may only be achieved by cells that have compensated; for example, alteration in the concentrations of components of intracellular signaling pathways may be essential for Itk-null cells to be selected for completion of maturation, but would result in reduced signaling in mature T cells.

The phenotypes that we observe in Itkdeficient mice may be parallel to some degree to the effect of Btk mutations in B cell development. In XLA patients, the number of bone marrow pre-B cells is normal, but there is impaired maturation to the B cell stage. This could be due to a developmental arrest or to exaggerated cell death at the pre-B to B-cell transition (Tsukada et al., 1993; Pearl et al., 1978). In xid mice, in which this effect is more subtle, it remains unclear why some B cells mature whereas oth- ers do not. Thymocytes in Itkdeficient mice appear to develop normally to the DP stage, but then display a defect in their transition to mature T cells. To better understand the function of Itk, it will be important to determine the nature of the mature T cells in the irk’- mice. An analysis of the TCR repertoire in mutant and control mice may shed some light on the mechanism by which Itk influences development of the immune system. It will also be important to determine whether these T cells are selected

Immunity 766

by a novel mechanism in the absence of Itk. Our studies using TCR transgenic mice in the Itk-deficient background have provided important clues to address these questions.

In summary, we have identified Itk as an important PTK involved in T cell develooment. Itk may be involved in reou- lation of the threshold of signal transduction during deiel- opment. One possibility is that Itk amplifies a positive signal from the TCR complex. In its absence, there would be a decrease in signal intensity, resulting in reduced efficiency of positive selection and reduced T cell proliferation upon TCR stimulation. Alternatively, Itk may deliver or am- plify an inhibitory signal. In its absence, there would be increased signal intensity, resulting in negative selection of cells that would otherwise undergo positive selection. The latter interpretation implies that mature T cells in if/r-‘- mice would have down-modulated the strength of signals to survive, which is consistent with their reduced proliferation upon TCR stimulation. Understanding the biochemical pathways in which Itk is involved will help to resolve these issues.

leaving a small portion of each to grow without feeder cells for DNA preparation as described (Laird et al., 1991).

PCR Analysis of ES Cells Screening of ES cells carrying homologous recombination was achieved by PCR as described previously (Stein et al., 1992; Soriano et al., 1991). ES cells from a pool of 6 clones or from a single clone were centrifuged and resuspended in 12 pl of the PCR lysis buffer (16.6 mM ammonium sulfate, 67mM Tris-HCI [pH 6.6],6.7mM MgCb, 5 mM 5-mercaptoethanol, 6.7 PM EDTA, 1.7 pM SDS, and 50 ug/ml proteinase K). The cells were lysed for 1 hr at 37OC followed by heat inactivation of proteinase K at 65OC for 10 min. The debris were spun down, and 5 ul of the supernatant was used for PCR reaction in a final volume of 25 pl. The reaction buffer for the PCR was as follows: 1 x PCR buffer (PCR lysis buffer without SDS and proteinase K), 1 mM each dNTP, 10% DMSO, 60 kg/ml bovine serum albumin (nuclease free), 0.1 pM each oligonucleotide primer, 2.5 U Taq polymerase (Boehringer Mannheim), and DNA provided by 5 ul heat- inactivated cell lysate. Usual PCR conditions were 93OC for 2 min, then 40 cycles at 93OC for 1 min, 56OC for 1 min. and 65OC for 5 min. using the DNA Thermal Cycler 4600 (Perkin Elmer). The oligo nucleotides used were the following: 3’neo, B’CTTGACGAGTTCTTC- TGAGGGGA-3’; andiUrPCRintron4-, 5%GGATGCCAGAATCAAClT- TGGTAG-3’.

Experimental Procedures

Construction of the Targeting Vector A modified version of the neo cassette, pgk(G)neopA, was obtained by swapping the Eagl to BamHl fragment of pKJ1 (Tybulewicz et al., 1991) with that of a new version of pMClneo Poly(A) (Stratagene), which contains the correct coding region of the neomycin phosphotransferase II gene (Yenofsky et al., 1990). The resulting pgk(G)neopA cassette contained the promoter and enhancer elements from the murine pgk-7 gene followed by the correct nsc coding region and the poly(A) addition site from the thymidine kinase gene (t/r) of the Herpes simplex virus (H. Boeuf, personal communication). The pgk(G)neopA cassette was subcloned into pBluescript KS(+) to generate pBSneo (hf. E. C. Crooks, personal communication).

it/r- mice were differentiated from if/r+- or irk+‘+ mice by Southern blot analysis. The 5’ probe was a 240 bp fragment internal to the putative intron 1. With the 5’ probe, the wild-type allele of itk was detected as a 25.1 kb Hindlll fragment and the mutant allele as a 17.6 kb Hindlll fragment. The 3’ probe was a 2.35 kb Hindlll-EcoRV fragment containing the putative introns 4 and 5 and exon 5. With the 3’probe, the wild-type allele of itk was detected as a 35 kb BamHl fragment and the mutant allele as a 3.65 kb BamHl fragment. Both probes are external to the targeting construct.

Expression of itk In COS Cells

A 400 bp Pvull-Pvull cDNA fragment encoding the N-terminal part (except the first 10 aa) of the murine Itk protein was used to screen a mouse genomic library in the 5DASH II vector (gift of Dr. P. Soriano, Fred Hutchison Cancer Center, Seattle, Washington). A stop codon was engineered at amino acid 76 of Itk within the putative exon 2 concomitant with an introduction of a Hindlll site. A 6.6 kb Xbal- Eco47lll fragment encompassing the mutated exon 2 was ligated onto the 5’end of pgk(G)neopA and a 1.9 kb Apal-Hindlll fragment internal to intron 4 of the itk gene was blunt-end ligated onto the 3’ end of pgk(G)neopA. The orientation of pgk(G)neopA was the same as the itk gene in the gene-targeting construct. The targeting vector was named pltkSTOP/KO/pgk(G)neopA, which was linearized with Notl before transfecting ES cells.

A 200 bp fragment identical to the murine itk cDNA was cloned by RT-PCR in the course of a screen that employed degenerate oligonucleotides to identify non-Src family PTKs from mouse thymus (Lai and Lemke, 1991). This fragment was used to screen a murine thymocyte cDNA library. A partial cDNA clone containing the 3’portion of the itk cDNA was obtained. The 5’ portion of the ifk cDNA was obtained by RT-PCR using ifk-specific oligonucleotides. The PCR fragment was

of the mur&ii cDNA(Siliciano et al., 1992; Heyeck and Berg, 1993). The full-length murine itk cDNA was then obtained by ligation of the 5’ portion with the 3’ portion. The full-length if/r cDNA was subcloned into pcDNAI/Amp (Invitrogen) via BamHl and Xhol to generate pCA.ifk. Transient transfection of pCAit& and control plasmids into COS7 cells was achieved using calcium phosphate-precipitated DNA in a standard protocol.

Immunopreclpltatlon and Western Blot Analysis Generation of ES Cells Carrying the Designed Ifk Mutatlon COS cells or thymocytes in a single cell suspension in phosphate- Jl ES cells (a gift of Drs. E. Li and R. Jaenisch) were maintained, buffered saline (PBS) were lysed with the addition of an equal volume transfected, and selected as described (Li et al., 1992; Crooks and of 2x RIPA buffer (1 x RIPA: 150 mM NaCI, 50 mM Tris-HCI [pH Littman, 1994). Selection was maintained for 10 days, after which 7.51. 10 mM NaF, 1% sodium deoxycholate, 1% NP-40, and 0.1% individual colonies were picked with a 20 ul micropipet. Each colony SDS) containing protease and phosphotase inhibitors (final concentra- was incubated in a V-shaped well of microtiter plates for 10 min at tion: leupeptin, 10 jtg/ml; pepstatin, 2 @ml; SBTI, 10 f&g/ml; PMSF, 37OC in 20 uI25 mM HEPES-buffered (pH 7.3) Hank’s Balanced Salt 2 mM; aprotinin, 50 &ml; sodium vanadate, 2.5 mM; and sodium Solution (without Caa or Mg-) containing 0.25% trypsin and 1 mM pyrophosphate, 6 mM) and incubating at 4OC for 20 min. The nuclei EDTA. Compl\te medium (200 ul) was added to each V-shaped well and cell debris were pelleted after spinning in a microfuge for 5 min. to terminate the trypsin digestion. 01 the dispersed ES cells, 160- Supernatants were collected and protein concentrations measured by 200 sl were transferred to each well of 24-well plates without G416 the Bradley method (Bio-Rad). Total cell lysates (200 ug) were loaded selection. of the remaining dispersed ES cells from the V-shaped on a 7.5% SDS-PAGE gel. Alternatively, the thymocyte lysates (406 wells, 5 pl were used for PCR analysis, initially in a pool with 5 other ug) were incubated for 1 hr at 4OC with ItkN4 (I:50 final), a polyclonal ES clones. After identifying a PCR-positive pool for homologous re- rabbit serum raised against a GST-fusion protein containing the combination at the ifk locus, each clone in the pool was analyzed by N-terminal region (amino acids l-175) of the mouse Itk protein. The PCR by taking 5 ftl again from the original cell suspension in the immune complex was precipitated by addition of 50 ul of 50% protein V-shaped wells. Only clones that gave a positive PCR signal for homol- A slurry in the lysis buffer. The immunoprecipitates were washed five ogous recombination were maintained in the 24-well plates by daily times with the lysis buffer and then boiled in 50 ul 2 x SDS sample change of medium. After 3 days, cells were harvested and frozen, loading dye containing 200 mM DTT before loading onto a 7.5% SDS-

Southern Blot Analysis

T Cell Phenotype in Ilk-Deficient Mice 787

PAGE gel. The gel was transferred to Immobilon-P membrane (Milli- pore) and incubated with ItkN4 (1:5000 in TBST with 5% nonfat dry milk) at room temperature for 1.5 hr. The secondary antibody (goat anti-rabbit-horseradish peroxidase. Boehringer Mannheim) was used at 1:20,000 followed by enhanced chemiluminescence detection of signals (Amersham). A different antiserum, ltkCL45, raised against a GST-fusion protein containing the C-terminal portion (amino acids 175-819) of the mouse Itk protein, gave similar results for Itk immunoblotting and immunoprecipitation (data not shown).

Flow Cytometric Analysis Flow cytometry was performed by staining 10 freshly isolated thymocytes and lymph node cells in 198 ul of PBS supplemented with 0.3% bovine serum albumin and 0.01% sodium azide on ice for 30 min. For three-color analysis, cells were first stained with various biotinconjugated antibodies against different lymphocyte surface proteins, followed by staining with a cocktail of streptavidin-TRICOLOR (Cal- tag), CD4-phycoerythrin (PE) (YTS191 .l, Caltag) and CDEfluores- cein isothiocyanate (FITC) (CT-CDBa, Caltag). Alternatively, lymph node cells were stained with CDS-PE (162C11, Pharmingen) and B220-FITC (RA3-862, Caltag). Cells were analyzed on a FACScan (Becton Dickinson) using the Lysis II software. Viable cells were gated on the basis of forward- and side-light scattering. Additional antibodies used were the following: anti-CD89 (H1.2F3), anti-heat stable antigen (M1/89), anti-TCRa8 (H57-597) anti-V83 (KJ25), anti-V88 (MR5-2) anti-Vall (RR8-1) and anti-IgM (R8-80.2) all from Pharmingen. Anti-H- Y-specific TCR (T3.70-biotin) was a gift of Dr. H.-S. Teh (University of British Columbia, Vancouver, Canada).

Cell Prollferatlon Assays The complete medium (RPM1 1840 supplemented with 10% fetal calf eerum, 2 mM L-glutamine, 50 uM j3-mercaptoethanol, 100 pM nones- sential amino acids, 50 U/ml penicillin and 50 &ml streptomycin) was used for cell preparation and culture. Responder cells were isolated from lymph nodes of littermates of if/r+‘- and iti’- mice (8-10 weeks old, H-Pm). CD4+ cells were purified by means of depleting other cell populations. In brief, a 10 cm tissue culture dish was coated overnight at 4OC with 5 ml of 5 &ml rabbit anti-mouse immunoglobulin in PBS (Zymed). Prior to use, the dish was washed five times with PBS, followed by blocking with 5 ml complete medium at room temperature for 10 min. B cells from about 2 x 10’ lymph node cells (in 5 ml) were depleted by two rounds of panning procedures, each consisting of a 30 min incubation at room temperature. To deplete CD8’ T cells and remaining B cells, nonadherent cells after panning were collected and resuspended in 0.3 ml complete medium ~1~~0.3 ml each of the hybridoma supernatant of Ml189 (rat anti-mouse HSA) and 538.72 (rat anti- mouse CD8a) for 30 min on ice. The cells were washed once and incubated with 200 ul of sheep anti-rat IgG-coated magnetic beads (Dynal) for 30 min on ice. The mixture was diluted and beads collected with a magnet. Cells remaining in suspension were harvested and found to contain typically 90% pure CD4+ T cells, which have not been inadvertently activated during preparation.

Splenocytes from BALBlc (H-2dM) and C57BU8 (H-2m) were inactivated by irradiation (about 2000 rads). Proliferation assays were set up in a volume of 200 ul in flat-bottomed wells of microtiter plates. For the MLR, serial dilution of 4 x 105, 2 x 105, 1 x 105, and 0.5 x 105 CD4+ cells were mixed with 5 x lo6 responder cells of various type in a well and incubated for 98 hr before pulsing for 24 hr with 1 uCi of PH]thymidine per well. For other cell proliferation assays, 5 x l(rCD4’cellsweremixed with5 x 105irradiatedC57BU8splenocytes plus anti-CD3 (purified 145-2C11, at 1.5,0.5, 0.187, and 0.058 &t/ml final concentration), or ConA (at 1, 0.5. 0.25, and 0.125 uglml final concentration), or 0.125 @ml ConA plus various concentration of human recombinant IL-2 (R and D). PMA (1 @ml) and ionomycin (500 nglml) were added to 5 x 10’ CD4+ cells either with or without irradiated C57BU8 splenocytes. Cells were incubated for 48 hr before pulsing for24 hrwith 1 pCiof[SH]thymidineperwell. Cellular radioactiv- ity was harvested using a microtiter plate cell harvester (Tomtec). All assays were done in triplicates and the mean and standard deviation are presented.

Acknowlsdgments

We thank E. Li and R. Jaenisch for providing the Jl ES cell line and for advice on its manipulation; P. Soriano and H.-S. Teh for providing the 129/Sv genomic librav and the biotinconjugated T3.70 antibody, respectively; N. Cacalano for providing GST-ltk fusion constructs; C. Lai and G. Lemke for the degenerate PTK oligonucleotides and S. Stuart and Jun Wu for the cloning of murine if/r cDNA; C. Davis for advice on flow cytometry; N. Killeen and C. Crooks for advice on ES cell culture and blastocyst injection; M. Hill for advice on Western blot and immunoprecipitation as well as for sharing hybridoma superna- tan& C. Crooks, K. Boyd, and J. Scarborough for help with mouse breeding and screening; L. Luo for help with the figures; A. Weiss, N. Killeen, C. Crooks, C. Davis, M. Hill, and N. van Ders for valuable discussions and critical comments. We would like to thank N. Killeen for providing space to allow completion of this work. X. C. L. is sup- ported by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation. D. R. L. is an investigator of the Howard Hughes Medical Institute.

Received July 21, 1995; revised September 22, 1995.

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Altered T cell receptor signaling and disrupted T cell development in mice lacking Itk

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