Rejection of syngeneic colon carcinoma by CTLs expressing single-chain antibody receptors codelivering CD28 costimulation

A new strategy to improve the therapeutic utility of redirected T cells for cancer involves the development of novel Ag-specific chimeric receptors capable of stimulating optimal and sustained T cell antitumor activity in vivo. Given that T cells require both primary and costimulatory signals for optimal activation and that many tumors do not express critical costimulatory ligands, modified single-chain Ab receptors have been engineered to codeliver CD28 costimulation. In this study, we have compared the antitumor potency of primary T lymphocytes expressing carcinoembryonic Ag (CEA)-reactive chimeric receptors that incorporate either TCR-zeta or CD28/TCR-zeta signaling. Although both receptor-transduced T cell effector populations demonstrated cytolysis of CEA(+) tumors in vitro, T cells expressing the single-chain variable fragment of Ig (scFv)-CD28-zeta chimera had a far greater capacity to control the growth of CEA(+) xenogeneic and syngeneic colon carcinomas in vivo. The observed enhanced antitumor activity of T cells expressing the scFv-CD28-zeta receptor was critically dependent on perforin and the production of IFN-gamma. Overall, this study has illustrated the ability of a chimeric scFv receptor capable of harnessing the signaling machinery of both TCR-zeta and CD28 to augment T cell immunity against tumors that have lost expression of both MHC/peptide and costimulatory ligands in vivo.     

T cell retargeting with MHC class I-restricted antibodies: the CD28 costimulatory domain enhances antigen-specific cytotoxicity and cytokine production

T cells require both primary and costimulatory signals for optimal activation. The primary Ag-specific signal is delivered by engagement of the TCR. The second Ag-independent costimulatory signal is mediated by engagement of the T cell surface costimulatory molecule CD28 with its target cell ligand B7. However, many tumor cells do not express these costimulatory molecules. We previously constructed phage display derived F(AB), G8, and Hyb3, Ab-based receptors with identical specificity but distinct affinities for HLA-A1/MAGE-A1, i.e., "TCR-like" specificity. These chimeric receptors comprised the FcepsilonRI-gamma signaling element. We analyzed whether linking the CD28 costimulation structure to it (gamma + CD28) could affect the levels of MHC-restricted cytolysis and/or cytokine production. Human scFv-G8(POS) T lymphocytes comprising the gamma + CD28 vs the gamma signaling element alone produced substantially more IL-2, TNF-alpha, and IFN-gamma in response to HLA-A1/MAGE-A1(POS) melanoma cells. Also a drastic increase in cytolytic capacity of scFv-G8(POS) T cells, equipped with gamma + CD28 vs the gamma-chain alone was observed.     

4-1BB is superior to CD28 costimulation for generating CD8+ cytotoxic lymphocytes for adoptive immunotherapy

Artificial APCs (aAPCs) genetically modified to express selective costimulatory molecules provide a reproducible, cost-effective, and convenient method for polyclonal and Ag-specific expansion of human T cells for adoptive immunotherapy. Among the variety of aAPCs that have been studied, acellular beads expressing anti-CD3/anti-CD28 efficiently expand CD4+ cells, but not CD8+ T cells. Cell-based aAPCs can effectively expand cytolytic CD8+ cells, but optimal costimulatory signals have not been defined. 4-1BB, a costimulatory molecule expressed by a minority of resting CD8+ T cells, is transiently up-regulated by all CD8+ T cells following activation. We compared expansion of human cytolytic CD8+ T cells using cell-based aAPCs providing costimulation via 4-1BB vs CD28. Whereas anti-CD3/anti-CD28 aAPCs mostly expand naive cells, anti-CD3/4-1BBL aAPCs preferentially expand memory cells, resulting in superior enrichment of Ag-reactive T cells which recognize previously primed Ags and efficient expansion of electronically sorted CD8+ populations reactive toward viral or self-Ags. Using HLA-A2-Fc fusion proteins linked to 4-1BBL aAPCs, 3-log expansion of Ag-specific CD8+ CTL was induced over 14 days, whereas similar Ag-specific CD8+ T cell expansion did not occur using HLA-A2-Fc/anti-CD28 aAPCs. Furthermore, when compared with cytolytic T cells expanded using CD28 costimulation, CTL expanded using 4-1BB costimulation mediate enhanced cytolytic capacity due, in part, to NKG2D up-regulation. These results demonstrate that 4-1BB costimulation is essential for expanding memory CD8+ T cells ex vivo and is superior to CD28 costimulation for generating Ag-specific products for adoptive cell therapy.     

Costimulatory receptors in a teleost fish: typical CD28, elusive CTLA4

T cell activation requires both specific recognition of the peptide-MHC complex by the TCR and additional signals delivered by costimulatory receptors. We have identified rainbow trout sequences similar to CD28 (rbtCD28) and CTLA4 (rbtCTLA4). rbtCD28 and rbtCTLA4 are composed of an extracellular Ig-superfamily V domain, a transmembrane region, and a cytoplasmic tail. The presence of a conserved ligand binding site within the V domain of both molecules suggests that these receptors likely recognize the fish homologues of the B7 family. The mRNA expression pattern of rbtCD28 and rbtCTLA4 in naive trout is reminiscent to that reported in humans and mice, because rbtCTLA4 expression within trout leukocytes was quickly up-regulated following PHA stimulation and virus infection. The cytoplasmic tail of rbtCD28 possesses a typical motif that is conserved in mammalian costimulatory receptors for signaling purposes. A chimeric receptor made of the extracellular domain of human CD28 fused to the cytoplasmic tail of rbtCD28 promoted TCR-induced IL-2 production in a human T cell line, indicating that rbtCD28 is indeed a positive costimulator. The cytoplasmic tail of rbtCTLA4 lacked obvious signaling motifs and accordingly failed to signal when fused to the huCD28 extracellular domain. Interestingly, rbtCTLA4 and rbtCD28 are not positioned on the same chromosome and thus do not belong to a unique costimulatory cluster as in mammals. Finally, our results raise questions about the origin and evolution of positive and negative costimulation in vertebrate immune systems.     

Intercellular adhesion molecule 1 is critical for activation of CD28-deficient T cells

Presentation of Ag to T lymphocytes in the absence of the requisite costimulatory signals leads to an Ag-specific unresponsiveness termed anergy, whereas Ag presentation in conjunction with costimulation leads to clonal expansion. B7/CD28 signaling has been shown to provide this critical costimulatory signal and blockade of this pathway may inhibit in vitro and in vivo immune responses. Although T cells from CD28-deficient mice are lacking in a variety of responses, they nonetheless are capable of various primary and secondary responses without the induction of anergy expected in the absence of costimulation. This suggests that there may be alternative costimulatory pathways that can replace CD28 signaling under certain circumstances. In this paper, we show that ICAM-1becomes a dominant costimulatory molecule for CD28-deficient T cells. ICAM-1 costimulates anti-CD3-mediated T cell proliferation and IL-2 secretion in CD28-deficient murine T cells. Furthermore, splenocytes from ICAM-1-deficient mice could not activate CD28-deficient T cells and splenocytes lacking both ICAM and CD28 fail to proliferate in response to anti-CD3-induced T cell signals. This confirms that not only can ICAM-1 act as a CD28-independent costimulator, but it is the dominant, requisite costimulatory molecule for the activation of T cells in the absence of B7/CD28 costimulation.     

Negative-feedback regulation of CD28 costimulation by a novel mitogen-activated protein kinase phosphatase, MKP6

TCR and CD28 costimulatory receptor-cooperative induction of T cell IL-2 secretion is dependent upon activation of mitogen-activated protein (MAP) kinases. Using yeast-hybrid technology, we cloned a novel CD28 cytoplasmic tail (CD28 CYT) interacting protein, MAP kinase phosphatase-6 (MKP6), which we demonstrate inactivates MAP kinases. Several lines of evidence indicate that MKP6 plays an important functional role in CD28 costimulatory signaling. First, in human peripheral blood T cells (PBT), expression of MKP6 is strongly up-regulated by CD28 costimulation. Second, transfer of dominant-negative MKP6 to PBT with the use of retroviruses primes PBT for the secretion of substantially larger quantities of IL-2, specifically in response to CD28 costimulation. A similar enhancement of IL-2 secretion is observed neither in response to TCR plus CD2 costimulatory receptor engagement nor in response to other mitogenic stimuli such as phorbol ester and ionomycin. Furthermore, this hypersensitivity to CD28 costimulation is associated with CD28-mediated hyperactivation of MAP kinases. Third, a retroviral transduced chimeric receptor with a CD28 CYT that is specifically unable to bind MKP6 costimulates considerably larger quantities of IL-2 from PBT than a similar transduced chimeric receptor that contains a wild-type CD28 CYT. Taken together, these results suggest that MKP6 functions as a novel negative-feedback regulator of CD28 costimulatory signaling that controls the activation of MAP kinases.     

CTLA-4 and CD28 mRNA are coexpressed in most T cells after activation. Expression of CTLA-4 and CD28 mRNA does not correlate with the pattern of lymphokine production.

Ag-presenting cells provide at least two distinct signals for T cell activation. T cell receptor-dependent stimulation is provided by presentation of a specific peptide Ag in association with MHC molecules. In addition, APC also supply costimulatory signals required for T cell activation that are neither Ag- nor MHC restricted. One such costimulatory signal is mediated via the interaction of B7 on APC with the CD28 receptor on T cells. Recently, CTLA-4 has been shown to be a second B7 receptor on T cells. In the present report, we have examined the expression of CD28 and CTLA-4 on a panel of resting and activated normal T cell subsets and T cell clones by RNA blot analysis in an attempt to determine whether their expression defines reciprocal or overlapping subsets. CD28 was detected in resting T cells, whereas CTLA-4 was not. After stimulation with PHA and PMA for 24 h, CTLA-4 mRNA was expressed in both the CD4+ and CD8+ subsets as well as in CD28+ T cells. We examined 37 human and six murine T cell clones that had been previously characterized for their cytokine production. After activation, CTLA-4 and CD28 mRNA were coexpressed in 36 of 37 human T cell clones and all six murine T cell clones. These included T cells of CD4+8-, CD4-8+, and CD4-8- phenotypes as well as clones with Th1 and Th2 cytokine profiles. In contrast, CD28 but not CTLA-4 mRNA was detected in leukemic T cell lines and myelomas. CTLA-4 and B7 mRNA but not CD28 mRNA was detected in two long term HTLV-I-transformed T cell lines. These data demonstrate that CD28 and CTLA-4 mRNA are coexpressed in most activated T cells and T cell clones, providing evidence that they do not define reciprocal subsets. Moreover, they are consistent with the hypothesis that B7 transmits its signal through a single receptor, CD28, on resting T cells, and multiple receptors, CD28 and CTLA-4, on activated T cells.     

OX40-mediated differentiation to effector function requires IL-2 receptor signaling but not CD28, CD40, IL-12Rbeta2, or T-bet

Ag-specific CD4 T cells transferred into unirradiated Ag-bearing recipients proliferate, but survival and accumulation of proliferating cells is not extensive and the donor cells do not acquire effector functions. We previously showed that a single costimulatory signal delivered by an agonist Ab to OX40 (CD134) promotes accumulation of proliferating cells and promotes differentiation to effector CD4 T cells capable of secreting IFN-gamma. In this study, we determined whether OX40 costimulation requires supporting costimulatory or differentiation signals to drive acquisition of effector T cell function. We report that OX40 engagement drives effector T cell differentiation in the absence of CD28 and CD40 signals. Two important regulators of Th1 differentiation, IL-12R and T-bet, also are not required for acquisition of effector function in CD4 T cells responsive to OX40 stimulation. Finally, we show that CD25-deficient CD4 T cells produce little IFN-gamma in the presence of OX40 costimulation compared with wild type, suggesting that IL-2R signaling is required for efficient OX40-mediated differentiation to IFN-gamma secretion.     

The cytoplasmic domain of CD28 is both necessary and sufficient for costimulation of interleukin-2 secretion and association with phosphatidylinositol 3''-kinase.

T-cell activation requires two signaling events. One is provided by the engagement of the T-cell antigen receptor, and the second represents a costimulatory signal provided by antigen-presenting cells. CD28 mediates a costimulatory signal by binding its ligands, B7-1 and B7-2, on antigen-presenting cells, but the signaling pathway activated by CD28 has not been identified. A homologous molecule, CTLA-4, expressed on activated T cells, also binds to B7-1 and B7-2, but whether it has a signaling function is not known. We performed a structure-function analysis of CD28 to identify the functional domain which activates signal transduction. Truncation of the 40-amino-acid CD28 cytoplasmic tail abrogated costimulatory signaling. Chimeric constructs containing the extracellular and transmembrane regions of CD8 linked to the cytoplasmic region of CD28 had a costimulatory signaling function. Similar chimeras containing the cytoplasmic tail of CTLA-4 did not signal. Thus, the cytoplasmic region of CD28, but not CTLA-4, is sufficient to mediate costimulatory signaling. In addition, after CD28 stimulation, the p85 subunit of phosphatidylinositol 3'-kinase and phosphatidylinositol 3'-kinase activity were found in CD28 immunoprecipitates. The CD8-CD28 chimera, which has a costimulatory signaling function, associates with p85, while the nonfunctioning CD8-CTLA-4 chimera and a CD8-zeta chimera do not associate with p85. These results suggest that phosphatidylinositol 3'-kinase is specifically activated by CD28 and may mediate proximal events in the costimulatory signaling pathway regulated by CD28.     

CD28-independent costimulation of T cells by OX40 ligand and CD70 on activated B cells.

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti-CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28- T cells from CD28-deficient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells.     

CD80 (B7-1) and CD86 (B7-2) are functionally equivalent in the initiation and maintenance of CD4+ T-cell proliferation after activation with suboptimal doses of PHA

Effective activation of T cells requires engagement of two separate T-cell receptors. The antigen-specific T-cell receptor (TCR) binds foreign peptide antigen-MHC complexes, and the CD28 receptor binds to the B7 (CD80/CD86) costimulatory molecules expressed on the surface of antigen-presenting cells (APC). The simultaneous triggering of these T-cell surface receptors with their specific ligands results in an activation of this cell. In contrast, CTLA-4 (CD152) is a distinct T-cell receptor that, upon binding to B7 molecules, sends an inhibitory signal to T cell activation. Many in vitro and in vivo studies demonstrated that both CD80 and CD86 ligands have an identical role in the activation of T cells. Recently, functions of B7 costimulatory molecules in vivo have been investigated in B7-1 and/or B7-2 knockout mice, and the authors concluded that CD86 could be more important for initiating T-cell responses, while CD80 could be more significant for maintaining these immune responses. In this study, we directly compared the role of CD80 and CD86 in initiating and maintaining proliferation of resting CD4(+) T cells in an in vitro mode system that allowed to provide the first signal-to-effector cells through the use of suboptimal doses of PHA and the second costimulatory signal through cells expressing CD80 or CD86, but not any other costimulatory molecules. Using this experimental system we demonstrate that the CD80 and CD86 molecules can substitute for each other in the initial activation of resting CD4(+) T cells and in the maintenance of their proliferative response.     

Differential utilization of CD134 as a functional receptor by diverse strains of feline immunodeficiency virus

The feline homologue of CD134 (fCD134) is the primary binding receptor for feline immunodeficiency virus (FIV), targeting the virus preferentially to activated CD4+ helper T cells. However, with disease progression, the cell tropism of FIV broadens such that B cells and monocytes/macrophages become significant reservoirs of proviral DNA, suggesting that receptor utilization may alter with disease progression. We examined the receptor utilization of diverse strains of FIV and found that all strains tested utilized CD134 as the primary receptor. Using chimeric feline x human CD134 receptors, the primary determinant of receptor function was mapped to the first cysteine-rich domain (CRD1) of fCD134. For the PPR and B2542 strains, the replacement of CDR1 of fCD134 (amino acids 1 to 64) with human CD134 (hCD134) alone was sufficient to confer nearly optimal receptor function. However, evidence of differential utilization of CD134 was revealed, since strains GL8, CPGammer (CPG41), TM2, 0827, and NCSU1 required determinants in the region spanning amino acids 65 to 85, indicating that these strains may require a more stringent interaction for infection to proceed.     

Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta /CD28 receptor.

Artificial receptors provide a promising approach to target T lymphocytes to tumor antigens. However, the receptors described thus far produce either an activation or a co-stimulatory signal alone, thus limiting the spectrum of functions accomplished by the genetically modified cells. Here we show that human primary T lymphocytes expressing fusion receptors directed to prostate-specific membrane antigen (PSMA) and containing combined T-cell receptor-zeta (TCRzeta), and CD28 signaling elements, effectively lyse tumor cells expressing PSMA. When stimulated by cell-surface PSMA, retrovirally transduced lymphocytes undergo robust proliferation, expanding by more than 2 logs in three weeks, and produce large amounts of interleukin-2 (IL-2). Importantly, the amplified cell populations retain their antigen-specific cytolytic activity. These data demonstrate that fusion receptors containing both TCR and CD28 signaling moieties are potent molecules able to redirect and amplify human T-cell responses. These findings have important implications for adoptive immunotherapy of cancer, especially in the context of tumor cells that fail to express major histocompatibility complex antigens and co-stimulatory molecules.     

CD28 regulates glucocorticoid-induced TNF receptor family-related gene expression on CD4+ T cells via IL-2-dependent mechanisms

The glucocorticoid-induced TNF-related gene receptor (GITR) is the newest member of the costimulatory molecule family and is expressed on both resting CD4+CD25+ regulatory T (T(R)) cells and activated CD4+ T cells. We investigated the endogenous mechanisms that regulate GITR expression on both T(R) and CD4+ T cells, as well as the functional interaction between GITR and other costimulatory molecules. CD28 stimulation increased GITR expression on both T(R) and CD4+ T cells via IL-2-dependent mechanisms. In addition, ligation of GITR and/or CD28 increased the level of CD4+ T cell proliferation and effector function under both APC-dependent and -independent conditions, suggesting that these costimulatory molecules cooperate to regulate CD4+ T cell activation and function by directly signaling to the CD4+ T cell. Thus, GITR may serve opposing functional roles on CD4+ T(R) and effector cells and alterations in GITR expression and/or function may tip the balance between immune tolerance and effector function.     

Analysis of the individual role of the TCRzeta chain in transgenic mice after conditional activation with chemical inducers of dimerization

Signaling through the TCR/CD3 complex plays a critical role in T-cell development and activation. Gene-targeted mice lacking particular components of this complex show arrested T-cell development in the thymus. As all TCR/CD3 components are required for efficient surface expression of the complex, it is difficult to assess the specific signaling role of each receptor component. To overcome this problem, we designed a strategy to examine the specific role(s) of individual receptor chains. A chimeric protein, containing binding domains for chemical inducers of dimerization fused to the cytoplasmic tail of TCRzeta, was generated. Activation of the chimeric receptor after stimulation with chemical dimerizers in Jurkat cells showed tyrosine phosphorylation of the TCRzeta chain chimera, recruitment of phosphorylated Zap70, and generation of NFAT in a reporter assay. Analysis of thymocytes from transgenic mice expressing this chimeric receptor showed that intracytoplasmic crosslinking of the chimera induced tyrosine phosphorylation of the protein, as well as a slow and very weak calcium mobilization response. However, this signaling did not lead to increased expression of activation markers, T-cell proliferation, or apoptosis. In addition, stimulation of thymocytes in suspension or in fetal thymic organ cultures with chemical inducers of dimerization did not lead to alterations in positive or negative selection. We conclude that signaling through the TCRzeta chain alone is not sufficient to generate downstream events leading to full T-cell activation or thymocyte selection; instead, additional CD3 components must be required to induce a functional response in primary thymocytes and peripheral T cells.     

Redirecting therapeutic T cells against myelin-specific T lymphocytes using a humanized myelin basic protein-HLA-DR2-zeta chimeric receptor

Therapies that Ag-specifically target pathologic T lymphocytes responsible for multiple sclerosis (MS) and other autoimmune diseases would be expected to have improved therapeutic indices compared with Ag-nonspecific therapies. We have developed a cellular immunotherapy that uses chimeric receptors to selectively redirect therapeutic T cells against myelin basic protein (MBP)-specific T lymphocytes implicated in MS. We generated two heterodimeric receptors that genetically link the human MBP84-102 epitope to HLA-DR2 and either incorporate or lack a TCRzeta signaling domain. The Ag-MHC domain serves as a bait, binding the TCR of MBP-specific target cells. The zeta signaling region stimulates the therapeutic cell after cognate T cell engagement. Both receptors were well expressed on primary T cells or T hybridomas using a tricistronic (alpha, beta, green fluorescent protein) retroviral expression system. MBP-DR2-zeta-, but not MBP-DR2, modified CTL were specifically stimulated by cognate MBP-specific T cells, proliferating, producing cytokine, and killing the MBP-specific target cells. The receptor-modified therapeutic cells were active in vivo as well, eliminating Ag-specific T cells in a humanized mouse model system. Finally, the chimeric receptor-modified CTL ameliorated or blocked experimental allergic encephalomyelitis (EAE) disease mediated by MBP84-102/DR2-specific T lymphocytes. These results provide support for the further development of redirected therapeutic T cells able to counteract pathologic, self-specific T lymphocytes, and specifically validate humanized MBP-DR2-zeta chimeric receptors as a potential therapeutic in MS.