The Src-family kinase, Fyn, regulates the activation of phosphatidylinositol 3-kinase in an interleukin 2-responsive T cell line

The proliferation of antigen-activated T cells is mediated by the T cell-derived growth factor, interleukin 2 (IL-2). The biochemical signaling cascades initiating IL-2-induced growth are dependent upon protein tyrosine kinase (PTK) activity. One IL-2-regulated PTK implicated in this cascade is the Src-family kinase, Fyn. Previous studies have described a physical association between Fyn and a potential downstream substrate, phosphatidylinositol 3-kinase (PI3- kinase) as well as the IL-2-dependent activation of PI3-kinase in T cells; however, the role of Fyn in IL-2-induced PI3-kinase activation remains unclear. In this report, we demonstrate that IL-2 stimulation triggers tyrosine phosphorylation of the p85 subunit of PI3-kinase in the murine T cell line, CTLL-2. Lysates prepared from growth factor- deprived and IL-2-stimulated T cells reconstituted both the binding of CTLL-2 cell-derived Fyn to and the IL-2-inducible tyrosine phosphorylation of exogenously added recombinant p85. Furthermore, overexpression of wild-type Fyn in these cells enhanced both the basal and IL-2-mediated activation of PI3-kinase. Additional studies of the Fyn-PI3-kinase interaction demonstrated that the Src homology 3 (SH3) domain of Fyn constitutes a direct binding site for the p85 subunit of PI3-kinase. These results support the notion that Fyn may be directly involved in the activation of the downstream signaling enzyme, PI3- kinase, in IL-2-stimulated T cells.     

Lymphokine-activated killer cells. Analysis of progenitors and effectors

IL-2 has been examined for its ability to regulate lymphokine-activated killer (LAK) activity. IL-2 is a potent activator of cytolytic activity against a wide array of tumor cells, including those from fresh autologous and allogeneic tumors. Using subpopulations of lymphoid cells that were separated on Percoll density gradients, and subsequently purified by immunoadsorbance, studies were performed to examine the phenotypes of progenitor and effector cells of human LAK cells and to compare them with the phenotype of activated NK cells. From these studies, it was evident that several lymphoid subsets, including CD3+, CDw16- and CD3-, CDw16+ cells could mediate LAK lysis of fresh tumor cells. Our examination of the kinetics of activation revealed that CDw16+, NKH1+ (NK-active) cells were maximally activated by 1-2 d. In contrast, CD3+ cells appeared not to achieve maximal cytolytic activity against fresh and cultured tumor cells until days 2-3. Using limiting-dilution frequency analysis, we showed that a large percentage of cytolytically active progenitors was present among the CDw16+, NKH1+ cells. The progenitor and effector cell frequencies appear to be 10-50 times higher in these populations compared to CD3+ cells. In addition, the selective blockage by mAb to the CD3 determinant of the T cell receptor complex indicated that these two effector cell phenotypes relied on different receptors to mediate their cytotoxic activity against tumor cells. Therefore, the accumulated data suggest that there is not a single unique progenitor of LAK activity, but rather that multiple subsets of lymphocytes become cytotoxic in response to IL-2. However, the NK cell population forms the largest single component of LAK cell activity in human peripheral blood.     

CD226 (DNAM-1) is involved in lymphocyte function-associated antigen 1 costimulatory signal for naive T cell differentiation and proliferation

Upon antigen recognition by the T cell receptor, lymphocyte function-associated antigen 1 (LFA-1) physically associates with the leukocyte adhesion molecule CD226 (DNAM-1) and the protein tyrosine kinase Fyn. We show that lentiviral vector-mediated mutant (Y-F322) CD226 transferred into naive CD4+ helper T cells (Ths) inhibited interleukin (IL)-12-independent Th1 development initiated by CD3 and LFA-1 ligations. Moreover, proliferation induced by LFA-1 costimulatory signal was suppressed in mutant (Y-F322) CD226-transduced naive CD4+ and CD8+ T cells in the absence of IL-2. These results suggest that CD226 is involved in LFA-1-mediated costimulatory signals for triggering naive T cell differentiation and proliferation. We also demonstrate that although LFA-1, CD226, and Fyn are polarized at the immunological synapse upon stimulation with anti-CD3 in CD4+ and CD8+ T cells, lipid rafts are polarized in CD4+, but not CD8+, T cells. Moreover, proliferation initiated by LFA-1 costimulatory signal is suppressed by lipid raft disruption in CD4+, but not CD8+, T cells, suggesting that the LFA-1 costimulatory signal is independent of lipid rafts in CD8+ T cells.     

Engagement of major histocompatibility complex class II molecules induces sustained, lymphocyte function-associated molecule 1-dependent cell adhesion

Antigenic stimulation is associated with enhanced adhesion between T cells and antigen-presenting cells (APC). Binding of ligands to the T cell antigen receptor activates the adhesion function of lymphocyte function-associated molecule 1 (LFA-1; CD11a/CD18). We demonstrate here that ligand binding to major histocompatibility complex class II (Ia) molecules also activates LFA-1 function, providing a reciprocal mechanism for the induction of adhesion between T cells and Ia+ APC. Adhesion was affected by a qualitative change in LFA-1 molecules and was reversed by the protein kinase C inhibitor sphingosine. These results define a novel role for Ia molecules as signal transducing receptors that regulate LFA-1-dependent adhesion via a putative, Ia-coupled protein kinase(s).     

LFA-1-specific therapy prolongs allograft survival in rhesus macaques

Outcomes in transplantation have been limited by suboptimal long-term graft survival and toxicities associated with current immunosuppressive approaches. T cell costimulation blockade has shown promise as an alternative strategy to avoid the side effects of conventional immunosuppressive therapies, but targeting CD28-mediated costimulation alone has proven insufficient to prevent graft rejection in primates. Donor-specific memory T (TM) cells have been implicated in costimulation blockade-resistant transplant rejection, due to their enhanced effector function and decreased reliance on costimulatory signaling. Thus, we have tested a potential strategy to overcome TM cell-driven rejection by targeting molecules preferentially expressed on these cells, such as the adhesion molecule lymphocyte function-associated antigen 1 (LFA-1). Here, we show that short-term treatment (i.e., induction therapy) with the LFA-1-specific antibody TS-1/22 in combination with either basiliximab (an IL-2Rα-specific mAb) and sirolimus (a mammalian target of rapamycin inhibitor) or belatacept (a high-affinity variant of the CD28 costimulation-blocker CTLA4Ig) prolonged islet allograft survival in nonhuman primates relative to control treatments. Moreover, TS-1/22 masked LFA-1 on TM cells in vivo and inhibited the generation of alloproliferative and cytokine-producing effector T cells that expressed high levels of LFA-1 in vitro. These results support the use of LFA-1-specific induction therapy to neutralize costimulation blockade-resistant populations of T cells and further evaluation of LFA-1-specific therapeutics for use in transplantation.     

Anti-Tumor CC49-zeta CD4 T cells possess both cytolytic and helper functions

The authors report that the nature of the T-cell-receptor--derived signal in normal CD4+ T cells can induce interleukin-2 (IL-2) secretion or perforin-mediated cytolytic activity. Normal human T cells were genetically modified to express the tumor antigen specific chimeric immune receptor, CC49-zeta. The CC49-zeta chimeric immune receptor is comprised of the intracellular signaling domains of the TCR CD3zeta protein fused to the single chain scFv of the humanized CC49 antibody, which binds the pan-adenocarcinoma tumor antigen TAG-72. Patient-specific T cells genetically modified to express the CC49-zeta receptor have been used in patients with colon cancer. The authors report that both CD4 and CD8 T cells expressing the CC49-zeta receptor mediated the major histocompatibility complex-unrestricted lysis of TAG-72--expressing tumor cells with comparable efficiency. However, although the CC49-zeta receptor mediated target cell lysis, it did not support the production of IL-2, even in the presence of CD28 stimulation. Robust IL-2 secretion and T-cell proliferation were observed when the same CD4 CC49-zeta T cells were stimulated through the CD28 receptor and endogenous T-cell receptor. These results indicate that CD4 T lymphocytes possess the capacity to act as both cytolytic and helper T cells and that this difference in effector function is controlled by the nature of the T-Cell receptor--derived signals.     

CD8(+) T cell tolerance to a tumor-associated antigen is maintained at the level of expansion rather than effector function

CD8+ T cell tolerance to self-proteins prevents autoimmunity but represents an obstacle to generating T cell responses to tumor-associated antigens. We have made a T cell receptor (TCR) transgenic mouse specific for a tumor antigen and crossed TCR-TG mice to transgenic mice expressing the tumor antigen in hepatocytes (gag-TG). TCRxgag mice showed no signs of autoimmunity despite persistence of high avidity transgenic CD8+ T cells in the periphery. Peripheral CD8+ T cells expressed phenotypic markers consistent with antigen encounter in vivo and had upregulated the antiapoptotic molecule Bcl-2. TCRxgag cells failed to proliferate in response to antigen but demonstrated cytolytic activity and the ability to produce interferon gamma. This split tolerance was accompanied by inhibition of Ca(2+) flux, ERK1/2, and Jun kinase phosphorylation, and a block in both interleukin 2 production and response to exogenous interleukin 2. The data suggest that proliferation and expression of specific effector functions characteristic of reactive cells are not necessarily linked in CD8+ T cell tolerance.     

Inhibition of human T cell activation by novel Src kinase inhibitors is dependent upon the complexity of the signal delivered to the cell

The activity of a novel series of protein tyrosine kinase inhibitors that are selective for the Src family has been assessed. The activity of these compounds [named CT-SKI (Celltech Src kinase inhibitors)] was investigated by assessing their potential to modulate T cell receptor activation, an event thought to involve the Src kinases Lck and Fyn. This series of compounds contained low-nanomolar inhibitors of Src kinases with selectivity over Csk, epidermal growth factor receptor kinase, protein kinase C, and zeta-associated 70-kDa protein. These compounds were shown to attenuate anti-CD3-induced T cell proliferation and block interleukin (IL)-2, IL-4, and interferon-gamma production, and CD25 expression in anti-CD3-activated T cells. In addition, inhibition of anti-CD3-induced, but not phorbol ester and calcium ionophore-induced IL-2 production, correlated with inhibition of in vitro Lck kinase activity. When more complex stimuli were used to activate T cells, as in the mixed lymphocyte reaction (MLR), these inhibitors proved to be less effective and inhibition of the MLR did not correlate with inhibition of isolated Lck enzyme. Interestingly, inhibition of anti-CD3-induced proliferation could be reversed by the addition of exogenous IL-2, indicating that signaling through the IL-2 receptor may not be critically dependent on any functional Src enzymes.     

Characterization of a novel subset of CD8(+) T cells that expands in patients receiving interleukin-12.

IL-12 has significant antitumor activity in mice that may be mediated by CD8(+) T cells. We show in this report that repeated subcutaneous injections of IL-12 in patients with cancer resulted in the selective expansion of a subset of peripheral blood CD8(+) T cells. This T cell subset expressed high levels of CD18 and upregulated IL-12 receptor expression after IL-12 treatment in vivo. In normal subjects, these CD3(+)CD8(+)CD18(bright) T cells expressed IL-12 and IL-2 receptors and adhesion/costimulatory molecules to a greater degree than other CD8(+) and CD4(+) T cells. They appeared morphologically as large granular lymphocytes, although they did not express NK cell markers such as CD56. In addition, CD8(+)CD18(bright) T cells were almost exclusively T cell receptor (TCR) alphabeta+, and exhibited a TCR Vbeta repertoire that was strikingly oligoclonal, whereas the Vbeta repertoire of CD18(dim) T cells was polyclonal. Although CD8+CD18(bright) T cells demonstrated little functional responsiveness to IL-12 or IL-2 alone in vitro, they responded to the combination of IL-12+IL-2 with strong IFN-gamma production and proliferation and enhanced non-MHC-restricted cytolytic activity. In contrast, CD18(dim) T cells were not activated by IL-12 or IL-2, alone or in combination. These findings demonstrate that CD8+CD18(bright) T cells are a unique population of peripheral blood lymphocytes with features of both memory and effector cells that are capable of TCR-independent activation through combined stimulation with IL-12+IL-2. As this activation results in IFN-gamma production and enhanced cytolytic activity, these T cells may play a role in innate as well as acquired immunity to tumors and infectious pathogens. Additional studies will be necessary to determine whether CD8+CD18(bright) T cells mediate the antitumor effect of IL-12 or IL-2 administered to cancer patients, and if so, whether maximal activation of these T cells with the combination of IL-12+IL-2 in vivo can augment the clinical effectiveness of these cytokines.     

Interleukin 2–mediated Uncoupling of T Cell Receptor α/β from CD3 Signaling

T cell activation and clonal expansion is the result of the coordinated functions of the receptors for antigen and interleukin (IL)-2. The protein tyrosine kinase p56^lck is critical for the generation of signals emanating from the T cell antigen receptor (TCR) and has also been demonstrated to play a role in IL-2 receptor signaling. We demonstrate that an IL-2–dependent, antigen-specific CD4⁺ T cell clone is not responsive to anti-TCR induced growth when propagated in IL-2, but remains responsive to both antigen and CD3ε-specific monoclonal antibody. Survival of this IL-2–dependent clone in the absence of IL-2 was supported by overexpression of exogenous Bcl-xL. Culture of this clonal variant in the absence of IL-2 rendered it susceptible to anti-TCR–induced signaling, and correlated with the presence of kinase-active Lck associated with the plasma membrane. The same phenotype is observed in primary, resting CD4⁺ T cells. Furthermore, the presence of kinase active Lck associated with the plasma membrane correlates with the presence of ZAP 70–pp21ζ complexes in both primary T cells and T cell clones in circumstances of responsive anti-TCR signaling. The results presented demonstrate that IL-2 signal transduction results in the functional uncoupling of the TCR complex through altering the subcellular distribution of kinase-active Lck.     

Activation of the COOH-terminal Src kinase (Csk) by cAMP-dependent protein kinase inhibits signaling through the T cell receptor

In T cells, cAMP-dependent protein kinase (PKA) type I colocalizes with the T cell receptor-CD3 complex (TCR/CD3) and inhibits T cell function via a previously unknown proximal target. Here we examine the mechanism for this PKA-mediated immunomodulation. cAMP treatment of Jurkat and normal T cells reduces Lck-mediated tyrosine phosphorylation of the TCR/CD3 zeta chain after T cell activation, and decreases Lck activity. Phosphorylation of residue Y505 in Lck by COOH-terminal Src kinase (Csk), which negatively regulates Lck, is essential for the inhibitory effect of cAMP on zeta chain phosphorylation. PKA phosphorylates Csk at S364 in vitro and in vivo leading to a two- to fourfold increase in Csk activity that is necessary for cAMP-mediated inhibition of TCR-induced interleukin 2 secretion. Both PKA type I and Csk are targeted to lipid rafts where proximal T cell activation occurs, and phosphorylation of raft-associated Lck by Csk is increased in cells treated with forskolin. We propose a mechanism whereby PKA through activation of Csk intersects signaling by Src kinases and inhibits T cell activation.     

Leukocyte function-associated antigen 1 is an activation molecule for human T cells

The leukocyte function-associated antigen 1 (LFA-1) molecule is well established as a surface protein involved in cellular adhesion and interaction, but there has been little information about whether engagement of this molecule can also directly modify cellular activation. These studies demonstrate that crosslinking the LFA-1 molecule on human T cell clones transmits a unique signal to the cell. Crosslinking LFA-1 alone did not increase intracellular calcium ([ CA2+]i), nor did crosslinking LFA-1 activate the cells as measured by IL-2 production or [3H]thymidine incorporation. However, when CD3 and LFA-1 were crosslinked, a more prolonged calcium signal was observed than when CD3 alone was crosslinked. Moreover, IL-2 production and DNA synthesis were greatly augmented. These responses could be demonstrated when LFA-1 was crosslinked via either the alpha or the beta chain, and required surface expression of the LFA-1 molecule as no enhancement was observed in T cell clones from a child with leukocyte adhesion deficiency. The enhancement of cellular activation by LFA-1 did not require that it be directly crosslinked to the CD3 complex. Thus, crosslinking LFA-1 alone with isotype-specific secondary antibodies on cells also pretreated with an anti-CD3 mAb of a different Ig isotype stimulated the cells as effectively as crosslinking both surface antigens with GaMIg. Similarly, a delayed, but sustained increase in [Ca2+]i was elicited. This increase in [Ca2+]i and the enhanced functional responses required engagement of CD3 with an intact bivalent anti-CD3 mAb, as crosslinking LFA-1 on cells also reacted with Fab fragments of an anti-CD3 mAb did not increase [Ca2+]i, nor activate the cells. These data indicate that LFA-1 can convey activation signals to T cells. Synergism in signaling can be observed upon crosslinking of LFA-1 and independently crosslinking CD3. In the physiologic interaction between T cells and accessory cells, the interaction of LFA-1 with its ligand, intercellular adhesion molecule 1, may therefore not only facilitate cellular adhesion, but also may amplify T cell activation by delivering costimulatory signals.     

Purified lymphocyte function-associated antigen 3 binds to CD2 and mediates T lymphocyte adhesion

CD2 is a T lymphocyte glycoprotein that functions in adhesion of T lymphocytes and also as a putative receptor for activation signals. Functional data suggest that LFA-3, a widely distributed cell surface glycoprotein, may be the biological ligand of CD2. We have purified LFA-3 from human erythrocytes and characterized the purified protein functionally. LFA-3 bound specifically to CD2+ cells, and this binding was inhibited by CD2 mAb. Conversely, purified LFA-3 inhibited binding of CD2 mAb to cells, and the concentration required for this effect suggests that LFA-3 half-saturated CD2 at 1-5 nM LFA-3. Purified LFA-3 inhibited rosetting of human and sheep erythrocytes with CD2+ T lymphoma cells and T lymphocytes, and mediated aggregation of a CD2+ T lymphoma cell line. Purified LFA-3 reconstituted into planar membranes mediated efficient CD2-dependent adhesion of T lymphoblasts. These data demonstrate that LFA-3 is a ligand for CD2 and that LFA-3 can mediate T lymphocyte adhesion.     

IL-11 separates graft-versus-leukemia effects from graft-versus-host disease after bone marrow transplantation

We recently showed that IL-11 prevents lethal graft-versus-host disease (GVHD) in a murine bone marrow transplantation (BMT) model of GVHD directed against MHC and minor antigens. In this study, we have investigated whether IL-11 can maintain a graft-versus-leukemia (GVL) effect. Lethally irradiated B6D2F1 mice were transplanted with either T cell-depleted (TCD) bone marrow (BM) alone or with BM and splenic T cells from allogeneic B6 donors. Animals also received host-type P815 mastocytoma cells at the time of BMT. Recipients were injected subcutaneously with recombinant human IL-11 or control diluent twice daily, from 2 days before BMT to 7 days after BMT. TCD recipients all died from leukemia by day 23. All control- and IL-11-treated allogeneic animals effectively rejected their leukemia, but IL-11 also reduced GVHD-related mortality. Examination of the cellular mechanisms of GVL and GVHD in this system showed that IL-11 selectively inhibited CD4-mediated GVHD, while retaining both CD4- and CD8-mediated GVL. In addition, IL-11 treatment did not affect cytolytic effector functions of T cells after BMT either in vivo or in vitro. Studies with perforin-deficient donor T cells demonstrated that the GVL effect was perforin dependent. These data demonstrated that IL-11 can significantly reduce CD4-dependent GVHD without impairing cytolytic function or subsequent GVL activity of CD8(+) T cells. Brief treatment with IL-11 shortly after BMT may therefore represent a novel strategy for separating GVHD and GVL.     

Monoclonal antibody directed against interleukin 2. I. Inhibition of T lymphocyte mitogenesis and the in vitro differentiation of alloreactive cytolytic T cells

Our recent studies have detailed the generation of B cell hybridomas whose IgG product significantly inhibits interleukin 2 (IL-2)-dependent T cell replication. Given the capacity of such hybridoma antibody to interfere with the activity of mouse, rat, and human IL-2, we asked whether anti-IL-2 IgG would mediate similar inhibitory effects on other in vitro immune responses. In this communication, we report that addition of purified anti-IL-2 monoclonal antibody to either mitogen- or alloantigen-stimulated spleen cells exerted markedly deleterious effects on both resultant T cell proliferation and the generation of cytolytic effector cells. These results provide serological evidence in support of the integral role that IL-2 plays in controlling antigen/mitogen-induced T cell proliferation and serves further to define the ability of monoclonal antibody against IL-2 to function as an immunosuppressive agent.     

Abrogating Cbl-b in effector CD8(+) T cells improves the efficacy of adoptive therapy of leukemia in mice

The clinical use of adoptive immunotherapy with tumor-reactive T cells to treat established cancers is limited in part by the poor in vivo survival and function of the transferred T cells. Although administration of exogenous cytokines such as IL-2 can promote T cell survival, such strategies have many nonspecific activities and are often associated with toxicity. We show here that abrogating expression of Casitas B-lineage lymphoma b (Cbl-b), a negative regulator of lymphocyte activation, in tumor-reactive CD8(+) T cells expanded ex vivo increased the efficacy of adoptive immunotherapy of disseminated leukemia in mice. Mechanistically, Cbl-b abrogation bypassed the requirement for exogenous IL-2 administration for tumor eradication in vivo. In addition, CD8(+) T cells lacking Cbl-b demonstrated a lower threshold for activation, better survival following target recognition and stimulation, and enhanced proliferative responses as a result of both IL-2-dependent and -independent pathways. Importantly, siRNA knockdown of Cbl-b in human CD8(+)CD28- effector T cell clones similarly restored IL-2 production and proliferation following target recognition independent of exogenous IL-2, enhanced IFN-γ production, and increased target avidity. Thus, abrogating Cbl-b expression in effector T cells may improve the efficacy of adoptive therapy of some human malignancies.