Immune Activation following Irbesartan Treatment in a Colorectal Cancer Patient: A Case Study.

Colorectal cancers are one of the most prevalent tumour types worldwide and, despite the emergence of targeted and biologic therapies, have among the highest mortality rates. The Personalized OncoGenomics (POG) program at BC Cancer performs whole genome and transcriptome analysis (WGTA) to identify specific alterations in an individual's cancer that may be most effectively targeted. Informed using WGTA, a patient with advanced mismatch repair-deficient colorectal cancer was treated with the antihypertensive drug irbesartan and experienced a profound and durable response. We describe the subsequent relapse of this patient and potential mechanisms of response using WGTA and multiplex immunohistochemistry (m-IHC) profiling of biopsies before and after treatment from the same metastatic site of the L3 spine. We did not observe marked differences in the genomic landscape before and after treatment. Analyses revealed an increase in immune signalling and infiltrating immune cells, particularly CD8+ T cells, in the relapsed tumour. These results indicate that the observed anti-tumour response to irbesartan may have been due to an activated immune response. Determining whether there may be other cancer contexts in which irbesartan may be similarly valuable will require additional studies.

Targeting the undruggable: immunotherapy meets personalized oncology in the genomic era.

Owing to recent advances in genomic technologies, personalized oncology is poised to fundamentally alter cancer therapy. In this paradigm, the mutational and transcriptional profiles of tumors are assessed, and personalized treatments are designed based on the specific molecular abnormalities relevant to each patient's cancer. To date, such approaches have yielded impressive clinical responses in some patients. However, a major limitation of this strategy has also been revealed: the vast majority of tumor mutations are not targetable by current pharmacological approaches. Immunotherapy offers a promising alternative to exploit tumor mutations as targets for clinical intervention. Mutated proteins can give rise to novel antigens (called neoantigens) that are recognized with high specificity by patient T cells. Indeed, neoantigen-specific T cells have been shown to underlie clinical responses to many standard treatments and immunotherapeutic interventions. Moreover, studies in mouse models targeting neoantigens, and early results from clinical trials, have established proof of concept for personalized immunotherapies targeting next-generation sequencing identified neoantigens. Here, we review basic immunological principles related to T-cell recognition of neoantigens, and we examine recent studies that use genomic data to design personalized immunotherapies. We discuss the opportunities and challenges that lie ahead on the road to improving patient outcomes by incorporating immunotherapy into the paradigm of personalized oncology.

Tumour-infiltrating FOXP3(+) lymphocytes are associated with cytotoxic immune responses and good clinical outcome in oestrogen receptor-negative breast cancer.

BACKGROUND: Regulatory T cells (Tregs) are commonly identified by expression of the transcription factor FOXP3 and are conventionally thought to promote cancer progression by suppressing anti-tumour immune responses. We examined the relationship between FOXP3(+) tumour-infiltrating lymphocytes (TIL) and prognosis in oestrogen receptor (ER)-negative breast cancer, a tumour subtype with poor clinical outcome in which TIL are abundant. METHODS: FOXP3(+) and CD8(+) TIL were assessed by immunohistochemistry in a cohort of 175 ER- breast tumours. Results were confirmed in an independent data set of 78 ER- breast tumours with publically available gene expression data. RESULTS: High FOXP3(+) TIL levels were strongly associated with prolonged recurrence-free survival (HR=0.461, P=0.0002), particularly among basal-like tumours (HR=0.280, P=0.0001), for which FOXP3 status was independent of standard prognostic factors. Over 75% of FOXP3(+) TIL in triple negative breast tumours displayed a conventional CD4(+)CD25(+) Treg phenotype. Importantly, FOXP3(+) TIL were positively correlated with CD8(+) (cytotoxic) T cells (r(s)=0.76, P<0.0001), and were prognostically insignificant in tumours with low levels of CD8(+) TIL. These observations were confirmed in an independent cohort. CONCLUSION: In contrast with current dogma, we show for the first time that FOXP3(+) TIL are associated with robust anti-tumour immunity and favourable prognosis in ER- breast cancer.

Conversion of ectoderm to mesoderm by cytoplasmic extrusion in leech embryos.

The role of cytoplasmic domains in the determination of the fates of ectodermal and mesodermal cells has been investigated in leech embryos. When yolk-deficient cytoplasm (teloplasm) was extruded from the animal pole of the zygote, the ectodermal precursor blastomere was converted to a mesodermal fate. This change of fate can be prevented by replacement of the extruded animal teloplasm with teloplasm from the vegetal pole. The fate of the mesodermal precursor blastomere was unaffected by teloplasm extrusion or rearrangement. These results demonstrate that ectodermal and mesodermal determination offate involves a binary decision dependent on the position of teloplasm along the animal-vegetal axis.

A membrane-proximal region of the interleukin-2 receptor gamma c chain sufficient for Jak kinase activation and induction of proliferation in T cells.

The interleukin-2 (IL-2) receptor (IL-2R) consists of three distinct subunits (alpha, beta, and gamma c) and regulates proliferation of T lymphocytes. Intracellular signalling results from ligand-mediated heterodimerization of the cytoplasmic domains of the beta and gamma c chains. To identify the residues of gamma c critical to this process, mutations were introduced into the cytoplasmic domain, and the effects on signalling were analyzed in the IL-2-dependent T-cell line CTLL2 and T-helper clone D10, using chimeric IL-2R chains that bind and are activated by granulocyte-macrophage colony-stimulating factor. Whereas previous studies of fibroblasts and transformed T cells have suggested that signalling by gamma c requires both membrane-proximal and C-terminal subdomains, our results for IL-2-dependent T cells demonstrate that the membrane-proximal 52 amino acids are sufficient to mediate a normal proliferative response, including induction of the proto-oncogenes c-myc and c-fos. Although gamma c is phosphorylated on tyrosine upon receptor activation and could potentially interact with downstream molecules containing SH2 domains, cytoplasmic tyrosine residues were dispensable for mitogenic signalling. However, deletion of a membrane-proximal region conserved among other cytokine receptors (cytoplasmic residues 5 to 37) or an adjacent region unique to gamma c (residues 40 to 52) abrogated functional interaction of the receptor chain with the tyrosine kinase Jak3. This correlated with a loss of all signalling events analyzed, including phosphorylation of the IL-2R beta-associated kinase Jak1, expression of c-myc and c-fos, and induction of the proliferative response. Thus, it appears in T cells that Jak3 is a critical mediator of mitogenic signaling by the gamma c chain.

New role for Shc in activation of the phosphatidylinositol 3-kinase/Akt pathway.

Most, if not all, cytokines activate phosphatidylinositol 3-kinase (PI-3K). Although many cytokine receptors have direct binding sites for the p85 subunit of PI-3K, others, such as the interleukin-3 (IL-3) receptor beta common chain (betac) and the IL-2 receptor beta chain (IL-2Rbeta), lack such sites, leaving the mechanism by which they activate PI-3K unclear. Here, we show that the protooncoprotein Shc, which promotes Ras activation by recruiting the Grb2-Sos complex in response to stimulation of cytokine stimulation, also signals to the PI-3K/Akt pathway. Analysis of Y-->F and "add-back" mutants of betac shows that Y577, the Shc binding site, is the major site required for Gab2 phosphorylation in response to cytokine stimulation. When fused directly to a mutant form of IL-2Rbeta that lacks other cytoplasmic tyrosines, Shc can promote Gab2 tyrosyl phosphorylation. Mutation of the three tyrosyl phosphorylation sites of Shc, which bind Grb2, blocks the ability of the Shc chimera to evoke Gab2 tyrosyl phosphorylation. Overexpression of mutants of Grb2 with inactive SH2 or SH3 domains also blocks cytokine-stimulated Gab2 phosphorylation. The majority of cytokine-stimulated PI-3K activity associates with Gab2, and inducible expression of a Gab2 mutant unable to bind PI-3K markedly impairs IL-3-induced Akt activation and cell growth. Experiments with the chimeric receptors indicate that Shc also signals to the PI-3K/Akt pathway in response to IL-2. Our results suggest that cytokine receptors lacking direct PI-3K binding sites activate Akt via a Shc/Grb2/Gab2/PI-3K pathway, thereby regulating cell survival and/or proliferation.

Biology of the interleukin-2 receptor.

Studies of the biology of the IL-2 receptor have played a major part in establishing several of the fundamental principles that govern our current understanding of immunology. Chief among these is the contribution made by lymphokines to regulation of the interactions among vast numbers of lymphocytes, comprising a number of functionally distinct lineages. These soluble mediators likely act locally, within the context of the microanatomic organization of the primary and secondary lymphoid organs, where, in combination with signals generated by direct membrane-membrane interactions, a wide spectrum of cell fate decisions is influenced. The properties of IL-2 as a T-cell growth factor spawned the view that IL-2 worked in vivo to promote clonal T-cell expansion during immune responses. Over time, this singular view has suffered from increasing appreciation that the biologic effects of IL-2R signals are much more complex than simply mediating T-cell growth: depending on the set of conditions, IL-2R signals may also promote cell survival, effector function, and apoptosis. These sometimes contradictory effects underscore the fact that a diversity of intracellular signaling pathways are potentially activated by IL-2R. Furthermore, cell fate decisions are based on the integration of multiple signals received by a lymphocyte from the environment; IL-2R signals can thus be regarded as one input to this integration process. In part because IL-2 was first identified as a T-cell growth factor, the major focus of investigation in IL-R2 signaling has been on the mechanism of mitogenic effects in cultured cell lines. Three critical events have been identified in the generation of the IL-2R signal for cell cycle progression, including heterodimerization of the cytoplasmic domains of the IL-2R beta and gamma(c) chains, activation of the tyrosine kinase Jak3, and phosphorylation of tyrosine residues on the IL-2R beta chain. These proximal events led to the creation of an activated receptor complex, to which various cytoplasmic signaling molecules are recruited and become substrates for regulatory enzymes (especially tyrosine kinases) that are associated with the receptor. One intriguing outcome of the IL-2R signaling studies performed in cell lines is the apparent functional redundancy of the A and H regions of IL-2R beta, and their corresponding downstream pathways, with respect to the proliferative response. Why should the receptor complex induce cell proliferation through more than one mechanism or pathway? One possibility is that this redundancy is an unusual property of cultured cell lines and that primary lymphocytes require signals from both the A and the H regions of IL-2R beta for optimal proliferative responses in vivo. An alternative possibility is that the A and H regions of IL-2R beta are only redundant with respect to proliferation and that each region plays a unique and essential role in regulating other aspects of lymphocyte physiology. As examples, the A or H region could prove to be important for regulating the sensitivity of lymphocytes to AICD or for promoting the development of NK cells. These issues may be resolved by reconstituting IL-2R beta-/-mice with A-and H-deleted forms of the receptor chain and analyzing the effect on lymphocyte development and function in vivo. In addition to the redundant nature of the A and H regions, there remains a large number of biochemical activities mediated by the IL-2R for which no clear physiological role has been identified. Therefore, the circumstances are ripe for discovering new connections between molecular signaling events activated by the IL-2R and the regulation of immune physiology. Translating biochemical studies of Il-2R function into an understanding of how these signals regulate the immune system has been facilitated by the identification of natural mutations in IL-2R components in humans with immunodeficiency and by the generation of mice with targeted mutations in these gen

Expression of chimeric granulocyte-macrophage colony-stimulating factor/interleukin 2 receptors in human cytotoxic T lymphocyte clones results in granulocyte-macrophage colony-stimulating factor-dependent growth.

Adoptive immunotherapy with ex vivo-expanded antigen-specific cytotoxic T lymphocytes (CTLs) has been shown to clear viral infections and eliminate tumors in murine models. Clinical trials have also reported promising data for the use of adoptive immunotherapy to treat cytomegalovirus (CMV) and Epstein-Barr viral (EBV) infections in bone marrow transplant recipients. For these indications, the need for ex vivo-expanded CTLs is often short lived, until the immune system is reconstituted by the donor transplant. In chronic disease settings, increased longevity of adoptively transferred CTLs and generation of memory will be necessary. The additional administration of helper functions normally supplied by antigen-specific T helper (Th) cells will probably be essential for long-term survival of adoptively transferred CTLs. Toward this goal of supplying helper functions, we transduced human CTLs with chimeric GM-CSFR/IL-2R receptors that deliver an IL-2 signal on binding GM-CSF. Clones expressing the chimeric receptors proliferated in response to GM-CSF. Stimulation with antigen induced GM-CSF production and resulted in an autocrine growth loop such that the CTL clones proliferated in the absence of exogenous cytokines. This type of genetic modification has potential for increasing the circulating half-life and, by extension, the efficacy of ex vivo-expanded CTLs.

A leech homolog of twist: evidence for its inheritance as a maternal mRNA.

In the development of leeches such as Helobdella robusta, mesodermal and ectodermal fates segregate to cells DM and DNOPQ, respectively, at fourth cleavage. As one step in identifying genes that may act in mesoderm determination, we have cloned the H. robusta homolog to the Drosophila gene twist. This homolog, designated Hro-twi, exhibits high (> 90%) amino acid identity with other twist-class genes within its basic-helix loop-helix (b-HLH) DNA binding motif and dimerization domain. Like twist, Hro-twi contains CAX-rich stretches: three stretches 5' to the b-HLH and one located 3' of the b-HLH motif. RT-PCR analysis suggests that Hro-twi is present throughout development, beginning as a maternal transcript in the oocyte.

MAGE-F1, a novel ubiquitously expressed member of the MAGE superfamily.

Most known members of the MAGE superfamily are expressed in tumors, testis and fetal tissues, which has been described as a cancer/testis or "CT" expression pattern. We have identified a novel member of this superfamily, MAGE-F1, which is expressed in all adult and fetal tissues tested. In addition to normal tissues, MAGE-F1 is expressed in many tumor types including ovarian, breast, cervical, melanoma and leukemia. MAGE-F1 is encoded on chromosome 3, identifying a sixth chromosomal location for a MAGE superfamily gene. The coding region of MAGE-F1 is contained within a single exon and includes a microsatellite repeat. Sequence analysis and expression profiles define a new class of ubiquitously expressed MAGE superfamily genes that includes MAGE-F1, MAGE-D1, MAGE-D2/JCL-1 and NDN. The finding that several MAGE genes are ubiquitously expressed suggests a role for MAGE encoded proteins in normal cell physiology. Furthermore, potential cross-reactivity to these ubiquitously expressed MAGE gene products should be considered in the design of MAGE-targeted immunotherapies for cancer.

Homodimerization of IL-2 receptor beta chain is necessary and sufficient to activate Jak2 and downstream signaling pathways.

Cytokine receptor signaling involves the Jak/Stat pathways. Heterotrimeric IL-2R (alpha, beta, gamma[c] chains) activates Jak1 and Jak3, whereas homodimeric PRLR activates Jak2. The requirements directing such specificity of Jak activation are unknown. We show that chimeric receptors containing the intracellular domain of IL-2Rbeta chain fused to the extracellular domain of either EPOR or Kit, a non-cytokine receptor, activate Jak2. This observation provides evidence that IL-2Rbeta intrinsically possesses the ability to activate Jak2, but that this property is only displayed in homodimerized complexes. Our data suggest a role for the stoichiometry of cytokine receptors in selective activation of Janus kinases.

Functional repair of transected spinal cord in embryonic chick.

The purpose of this study was to determine the developmental stage of the chick embryo when descending spinal tracts lose the capacity for anatomical and functional repair after complete transection of the thoracic spinal cord. Previous studies have demonstrated that the first reticulospinal projections descend to the lumbar cord by embryonic day (E) 5. A comparison of the distribution and density of retrogradely labelled brainstem-spinal neurons in embryos versus hatchling chicks suggests that the descent of all brainstem-spinal projections is essentially complete to lumbar levels between E10 and El2. Transections and control sham operations were performed on different embryos from E3 through E14 of development. After a recovery period of 5-18 days, the extent of anatomical repair was assessed by injecting a small volume of a retrograde tract-tracing chemical into the upper lumbar spinal cord, caudal to the transection site. The brainstem nuclei were then examined for the number and distribution of retrogradely labelled brainstem-spinal neurons. In comparison to control animals, anatomical recovery appeared to be complete for embryos transected as late as E12, whereas thoracic cord transections conducted on E13-E14 resulted in reduced labelling of most brainstem-spinal nuclei. In addition, a number of E3-E6 transected embryos were allowed to hatch and with some assistance a few E7-E14 transected embryos also hatched. Functional recovery was assessed by behavioral observations and by focal electrical stimulation of brainstem locomotor regions (known to have direct projections to the lumbar spinal cord). Brainstem stimulation experiments were undertaken on transected and control embryos, either in ovo on E18-E20 or after hatching. Leg and wing muscle electromyographic recordings were used to monitor any brainstem evoked motor activity. Voluntary open-field locomotion (hatchling chicks) or brainstem evoked locomotion (embryonic or hatchling) in animals transected on or before E12 was indistinguishable from that observed in control (i.e. sham-operated or unoperated) chicks, indicating that complete functional recovery had occurred. In contrast, chicks transected on or after El3 showed reduced functional recovery. Since a previous study has shown that neurogenesis in chick brainstem-spinal neurons is complete prior to E5, the possible intrinsic neuronal mechanisms underlying the repair of descending supraspinal pathways are: (1) subsequent projections from later developing (undamaged) neurons, or (2) regrowth of previously axotomized projections (regeneration). For the E5-E12 chick embryos examined in this study, significant descending supraspinal fibers are present within the thoracic cord at the time of transection. Even if the transection is made at E12, when descending projections have completed their development to the lumbar cord, there is still a similar number and distribution of brainstem-spinal neurons labelled afterward (when compared to controls). This suggests that regeneration of previously axotomized projections may account for some of the observed anatomical and functional repair of brainstem-spinal pathways.

Cytoplasmic and cortical determinants interact to specify ectoderm and mesoderm in the leech embryo.

In leech embryos, segmental ectoderm and mesoderm are produced by a pair of sister cells located near the animal and vegetal poles, respectively. We have investigated the mechanism that localizes ectodermal and mesodermal fates along the animal-vegetal axis. The results of cleavage arrest and cell ablation experiments suggest that the full range of normal cell interactions are not required for this process. However, when the animal and vegetal hemispheres are separated by re-orientation of the first cleavage plane from meridional to equatorial, the ectodermal fate co-segregates with the animal hemisphere and the mesodermal fate with the vegetal hemisphere. Two pools of yolk-deficient cytoplasm, called teloplasm, are located at the animal and vegetal poles of the zygote, but separation of the animal and vegetal teloplasms is not sufficient for the segregation of ectodermal and mesodermal fates. Rather, complete segregation of fates requires an equatorial cleavage orientation that separates not only the two teloplasms, but also the animal and vegetal cortical regions. This, in conjunction with previous findings, indicates that ectodermal determinants are localized to the cell cortex in the animal hemisphere of the zygote. We propose that these determinants segregate to the ectodermal precursor and interact with factors in teloplasm to transform the fate of this cell from a mesodermal ground state to ectoderm.

Phosphatidylinositol 3-kinase potentiates, but does not trigger, T cell proliferation mediated by the IL-2 receptor.

Proliferative signaling by the IL-2R can occur through two distinct pathways, one mediated by Stat5 and one by the adaptor protein Shc. Although Stat5 induces T cell proliferation by serving as a transcription factor, the mechanism of proliferative signaling by Shc is poorly defined. We examined the roles of two major signaling pathways downstream of Shc, the p44/p42 mitogen-activated protein kinase (extracellular signal-related kinase (Erk)) and phosphatidylinositol 3-kinase (PI3K) pathways, in promitogenic gene induction and proliferation in the IL-2-dependent T cell line CTLL-2. Using IL-2R mutants and specific pharmacologic inhibitors, we found that the PI3K, but not Erk, pathway is required for maximal induction of c-myc, cyclin D2, cyclin D3, cyclin E, and bcl-x(L) by Shc. To test whether the PI3K pathway is sufficient for proliferative signaling, a tamoxifen-regulated form of PI3K (mp110*ER) was expressed in CTLL-2 cells. Activation of the PI3K pathway through mp110*ER failed to up-regulate expression of the c-myc, cyclin D2, cyclin D3, cyclin E, bcl-2, or bcl-x(L) genes or down-regulate expression of p27(Kip1), even when coactivated with the Janus kinases (Jak) or the Raf/Erk pathway. Moreover, mp110*ER induced modest levels of thymidine incorporation without subsequent cell division. Although insufficient for mitogenesis, mp110*ER enhanced Stat5-mediated proliferative signaling through a mechanism independent of Stat5 transcriptional activity. Thus, in addition to serving a necessary, but insufficient role in Shc-mediated promitogenic gene expression, the PI3K pathway contributes to T cell proliferation by potentiating mitogenic signaling by Stat5.

Cytoplasmic domains of the interleukin-2 receptor beta and gamma chains mediate the signal for T-cell proliferation.

The interleukin-2 receptor (IL-2R) consists of three distinct chains (alpha, beta, gamma) which bind IL-2 and generate a proliferative signal in T cells. To define the mechanism of receptor activation, chimaeric receptors were constructed from the intracellular region of either IL-2R beta or IL-2R gamma and the extracellular region of c-kit, a receptor tyrosine kinase that homodimerizes on binding stem cell factor (SCF). We report here that binding of SCF to the beta-chain chimaera induced proliferation of the pro-B-cell line BA/F3, but not T cells. But in T cells expressing both the beta- and gamma-chain chimaeras, SCF induced proliferation and tyrosine phosphorylation characteristic of the native IL-2R signal. Chimaeric IL-2 receptor beta and gamma chains constructed with the heterodimeric extracellular regions of the granulocyte-macrophage colony stimulating factor receptor (GM-CSFR) also provided the IL-2R signal. Thus, heterodimerization of the cytoplasmic domains of IL-2R beta and -gamma appears necessary and sufficient for signalling in T cells.

Effect of osteochondral defects on articular cartilage. Contact pressures studied in dog knees.

Full thickness osteochondral defects 6 mm in diameter were created in the weight-bearing regions of the femoral condyles in 5 adult mongrel dogs to study the contact pressure changes accompanying healing. A digital imaging technique employing Fuji Prescale film mapped contact pressures following 11 months of healing for comparison with contralateral normal knees, and knees with freshly made defects. Although all defects healed uneventfully with subchondral plate reconstitution and with growth restoration of the articular surface, the repair soft tissue appeared histologically to be primarily fibrous tissue, with varying degrees of a fibrocartilaginous component. The mean and peak stresses about fresh defects were not appreciably different from those about healed defects. Neither were there substantial differences in the total cartilage area making contact, except when very low loads were applied. The results suggest that the repair tissue is of poor mechanical quality, and does not contribute appreciably to weight bearing. The cartilage adjacent to the defect did not experience high stresses; neither gross nor light microscopic evidence of degeneration appeared at 11 months. If degeneration does occur following such defects, our data suggest that it is not because of elevated contact stresses.

The IL-4 receptor alpha-chain cytoplasmic domain is sufficient for activation of JAK-1 and STAT6 and the induction of IL-4-specific gene expression.

The common gamma-chain (gamma(c)) is a functional component of the IL-4R, yet cells lacking gamma(c) are able to respond to IL-4. This has led to the suggestion that a surrogate gamma'-chain, which can interact with the IL-4R alpha chain to mediate signaling, is expressed on cells lacking gamma(c). An alternative possibility is that in the absence of gamma(c), the IL-4R alpha chain is able to transduce signals by homodimerization. To test this latter possibility, a chimeric receptor containing the extracellular domain of c-kit (the stem cell factor (SCF) receptor) and the cytoplasmic and transmembrane domains of the IL-4R alpha chain was generated. Treatment of cells expressing the chimeric receptor kit/IL-4R alpha with SCF induces activation of the IL-4R alpha-associated kinase JAK-1 and the transcription factor STAT6. However, tyrosine phosphorylation of JAK-3, which associates with gamma(c), is not induced by SCF in these cells. SCF-mediated ligation of kit/IL-4R alpha is sufficient to elicit IL-4-specific gene expression, including up-regulation of CD23 and synthesis of germ-line epsilon transcripts. In the T cell line CTLL2, ligation of kit/IL-4R alpha induces cellular proliferation. Finally, in JAK-1-deficient HeLa cells, STAT6 activation by IL-4 is completely abolished. Together, these data demonstrate that the IL-4R alpha cytoplasmic domain is sufficient to activate JAK-1 and STAT6 and to induce expression of IL-4 target genes, thus identifying a mechanism by which IL-4 signaling can proceed in the absence of JAK-3 and gamma(c).

Requirement for an initial signal from the membrane-proximal region of the interleukin 2 receptor gamma(c) chain for Janus kinase activation leading to T cell proliferation.

The interleukin 2 receptor (IL-2R) generates proliferative signals in T lymphocytes by ligand-induced heterodimerization of two chains, IL-2Rbeta and gamma(c), which associate with the tyrosine kinases Jak1 and Jak3, respectively. Genetic and molecular studies have demonstrated that Jak3 is essential for mitogenic signaling by the gamma(c) chain; because it is also the only molecule known to associate with gamma(c), we speculated that Jak3 might be sufficient for signaling by this chain. Therefore, fusion proteins were constructed in which all or part of the cytoplasmic domain of gamma(c) was replaced by Jak3. Signaling was evaluated in the IL-2-dependent T cell line CTLL-2 using chimeric IL-2Rbeta and gamma(c) chains that bind and are activated by the cytokine granulocyte-macrophage colony-stimulating factor. Chimeric gamma(c) chains containing only Jak3 in the cytoplasmic domain failed to mediate proliferation of CTLL-2 cells, but addition of a conserved membrane-proximal (PROX) domain of gamma(c) in tandem with Jak3 fully reconstituted gamma(c) function. The requirement for the PROX domain reflected an essential role in the activation of Jak3 in vivo. Despite lacking defined catalytic motifs, PROX induced an early Jak-independent signal, including tyrosine phosphorylation of IL-2Rbeta and the tyrosine phosphatase SHP-2. The results define the minimal signaling components of gamma(c) and suggest a new mechanism by which the IL-2R initiates signaling in response to ligand.

The IL-2 receptor promotes lymphocyte proliferation and induction of the c-myc, bcl-2, and bcl-x genes through the trans-activation domain of Stat5.

Studies assessing the role of Stat5 in the IL-2 proliferative signal have produced contradictory, and thus inconclusive, results. One factor confounding many of these studies is the ability of IL-2R to deliver redundant mitogenic signals from different cytoplasmic tyrosines on the IL-2R beta-chain (IL-2Rbeta). Therefore, to assess the role of Stat5 in mitogenic signaling independent of any redundant signals, all cytoplasmic tyrosines were deleted from IL-2Rbeta except for Tyr510, the most potent Stat5-activating site. This deletion mutant retained the ability to induce Stat5 activation and proliferation in the T cell line CTLL-2 and the pro-B cell line BA/F3. A set of point mutations at or near Tyr510 that variably compromised Stat5 activation also compromised the proliferative signal and revealed a quantitative correlation between the magnitude of Stat5 activation and proliferation. Proliferative signaling by a receptor mutant with a weak Stat5 activating site could be rescued by overexpression of wt Stat5a or b. Additionally, the ability of this receptor mutant to induce c-myc, bcl-x, and bcl-2 was enhanced by overexpression of wt Stat5. By contrast, overexpression of a version of Stat5a lacking the C-terminal trans-activation domain inhibited the induction of these genes and cell proliferation. Thus, Stat5 is a critical component of the proliferative signal from Tyr510 of the IL-2R and regulates expression of both mitogenic and survival genes through its trans-activation domain.