CD28, IL-2-independent costimulatory pathways for CD8 T lymphocyte activation.

We investigate, here, the mechanism of the costimulatory signals for CD8 T cell activation and confirm that costimulation signals via CD28 do not appear to be required to initiate proliferation, but provide survival signals for CD8 T cells activated by TCR ligation. We show also that IL-6 and TNF-alpha can provide alternative costimulatory survival signals. IL-6 and TNF-alpha costimulate naive CD8 T cells cultured on plate-bound anti-CD3 in the absence of CD28 ligation. They act directly on sorted CD8-positive T cells. They also costimulate naive CD8 T cells from Rag-2-deficient mice, bearing transgenic TCRs for HY, which lack memory cells, a potential source of IL-2 secretion upon activation. IL-6 and TNF-alpha provide costimulation to naive CD8 T cells from CD28, IL-2, or IL-2Ralpha-deficient mice, and thus function in the absence of the B7-CD28 and IL-2 costimulatory pathways. The CD8 T cell generated via the anti-CD3 plus IL-6 and TNF-alpha pathway have effector function in that they express strong cytolytic activity on Ag-specific targets. They secrete only very small amounts of any of the cytokines tested upon restimulation with peptide-loaded APC. The ability of the naive CD8 T cells to respond to TCR ligation and costimulatory signals from IL-6 and TNF-alpha provides a novel pathway that can substitute for signals from CD4 helper cells or professional APC. This may be significant in the response to viral Ags, which can be potentially expressed on the surface of any class I MHC-expressing cell.     

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.     

NKG2D is a costimulatory receptor for human naive CD8+ T cells

In humans, all alpha beta CD8+ T cells express NKG2D, but in mouse, it is only expressed by activated and memory CD8+ T cells. We purified human naive CD8+ T cells to show that NKG2D serves as a costimulatory receptor for TCR induced Ca2+ mobilization and proliferation. The resulting effector cells are skewed toward a type 1 phenotype and produce high levels of IFN-gamma and TNF-alpha. NKG2D ligands, MHC class I chain-related (MIC)A, MICB, and UL16-binding proteins are expressed on the proliferating cells and NKG2D is down-regulated. The addition of the homeostatic cytokines IL-7 and IL-15 to the culture medium not only enhances proliferation but also counteracts the down-regulation of NKG2D, more so than the addition of IL-2. These results indicate that NKG2D can regulate the priming of human naive CD8+ T cells, which may provide an alternative mechanism for potentiating and channeling the immune response.     

Differential costimulatory effects of adhesion molecules B7, ICAM-1, LFA-3, and VCAM-1 on resting and antigen-primed CD4+ T lymphocytes.

Optimal proliferation of T cells although initiated via ligation of the CD3/TCR complex requires additional stimulation resulting from adhesive interactions between costimulatory receptors (R) on T cells and their counter-R on APC. At least four distinct adhesion molecules (counter-R) present on APC, B7, ICAM-1 (CD54), LFA-3 (CD58), and VCAM-1 have been individually shown to costimulate T cell activation. Because some of these molecules may be expressed simultaneously on APC, it has been difficult to examine relative contributions of individual counter-R during the induction of T cell proliferation. We have produced soluble IgC gamma 1 fusion chimeras (receptor globulins or Rg) of B7, ICAM-1, LFA-3, and VCAM-1 and compared their relative abilities to costimulate proliferation of resting or Ag-primed CD4+ T cells. When co-immobilized with mAb directed at TCR alpha beta or CD3 but not CD2 or CD28, each Rg induced proliferation of both resting and Ag-primed CD4+ cells. In contrast, similarly co-immobilized CD7 Rg or ELAM-1 Rg were ineffective. Resting CD4+ T cells produced more IL-2, expressed significantly higher levels of IL-2R alpha, and proliferated more efficiently when costimulated with either ICAM-1 Rg or VCAM-1 Rg than with B7 Rg or LFA-3 Rg. CD4+ CD45RO+ memory T cells proliferated more vigorously in response to the costimulation by each of the four Rg than CD4+ CD45RA+ naive T cells. In contrast with the behavior of resting CD4+ T cells, proliferation of Ag-preactivated CD4+ T cells was most efficient when costimulated by B7 Rg. The costimulatory effect of LFA-3 Rg on Ag-primed CD4+ T cells was weaker than that of B7 Rg but was significantly greater than that of either ICAM-1 Rg or VCAM-1 Rg. These results suggest that resting and Ag-primed CD4+ T cells preferentially respond by proliferation to different costimulatory counter-R. ICAM-1 and VCAM-1 may be involved in the initiation of proliferation of Ag-responsive T cells, and B7 and LFA-3 may facilitate sustained proliferation of Ag-primed T cells. The cumulative costimulation by the above counter-R may facilitate optimal expression of various regulatory and effector functions of T cells.     

NKG2D receptors induced by IL-15 costimulate CD28-negative effector CTL in the tissue microenvironment.

Unlike primary T cells in lymph nodes, effector CD8(+) CTL in tissues do not express the costimulatory receptor CD28. We report that NKG2D, the receptor for stress-induced MICA and MICB molecules expressed in the intestine, serves as a potent costimulatory receptor for CTL freshly isolated from the human intestinal epithelium. Expression and function of NKG2D are selectively up-regulated by the cytokine IL-15, which is released by the inflamed intestinal epithelium. These findings identify a novel CTL costimulatory pathway regulated by IL-15 and suggest that tissues can fine-tune the activation of effector T cells based on the presence or absence of stress and inflammation. Uncontrolled secretion of IL-15 could lead to excessive induction of NKG2D and thus contribute to the development of autoimmune disease by facilitating the activation of autoreactive T cells.     

Choice of resident costimulatory molecule can influence cell fate in human naïve CD4+ T cell differentiation

With antigen stimulation, naïve CD4+ T cells differentiate to several effector or memory cell populations, and cytokines contribute to differentiation outcome. Several proteins on these cells receive costimulatory signals, but a systematic comparison of their differential effects on naïve T cell differentiation has not been conducted. Two costimulatory proteins, CD28 and ICAM-1, resident on human naïve CD4+ T cells were compared for participation in differentiation. Under controlled conditions, and with no added cytokines, costimulation through either CD3+CD28 or CD3+CAM-1 induced differentiation to T effector and T memory cells. In contrast, costimulation through CD3+ICAM-1 induced differentiation to Treg cells whereas costimulation through CD3+CD28 did not.     

CD99 costimulation up-regulates T cell receptor-mediated activation of JNK and AP-1

Although CD28 is the principal T cell costimulatory molecule for the T cell receptor, a number of other cell surface proteins have costimulatory functions and perform specific roles in different contexts. Here we analyzed the mechanism of CD99 costimulation of the T cell receptor. Cooperation of CD99 engagement with suboptimal TCR/CD3 signals resulted in greatly enhanced CD4+ T cell proliferation. CD99 costimulation also led to elevated expression of CD25 and GM1 on the CD4+ T cell surface within 3 days. In Jurkat TAg cells, CD99 costimulation led to increased apoptosis compared to stimulation with CD3 or CD99 alone. CD99 costimulation also augmented activation of MAP kinases, especially of JNK, and increased AP-1 activation was also observed using a luciferase reporter assay. These results show that CD99 has a costimulatory function for T cells and acts by a mechanism distinct from CD28.     

The role of intercellular adhesion molecule-1/LFA-1 interactions in the generation of tumor-specific CD8+ T cell responses

The activation of naive CD4+ T cells requires both TCR engagement and a second costimulatory signal mediated by the interaction of CD28 with CD80/CD86 expressed on professional APC. However, the situation for naive CD8+ T cells is less clear. Although evidence indicates that induction of CD8+ T cell responses is also dependent on professional APC, the ability of some tumors, which do not express CD80/CD86, to induce CTL suggests that other pathways of costimulation exist for the activation of CD8+ T cells. We examined the ability of tumor cells expressing different levels of a tumor-specific Ag to directly prime CD8+ T cells. We demonstrate that CD8+ T cells are directly activated by tumor cells in a CD80/CD86-CD28 independent manner. In this system, costimulation requires ICAM-1/LFA-1 interaction. This results in the generation of CTL capable of inhibiting tumor growth in vivo, and maintaining long-term survival.     

Naive CD8+ T cells do not require costimulation for proliferation and differentiation into cytotoxic effector cells

Most current models of T cell activation postulate a requirement for two distinct signals. One signal is delivered through the TCR by engagement with peptide/MHC complexes, and the second is delivered by interaction between costimulatory molecules such as CD28 and its ligands CD80 and CD86. Soluble peptide/MHC tetramers provide an opportunity to test whether naive CD8+ T cells can be activated via the signal generated through the TCR-alphabeta in the absence of any potential costimulatory molecules. Using T cells from two different TCR transgenic mice in vitro, we find that TCR engagement by peptide/MHC tetramers is sufficient for the activation of naive CD8+ T cells. Furthermore, these T cells proliferate, produce cytokines, and differentiate into cytolytic effectors. Under the conditions where anti-CD28 is able to enhance proliferation of normal B6 CD4+, CD8+, and TCR transgenic CD8+ T cells with anti-CD3, we see no effect of anti-CD28 on proliferation induced by tetramers. The results of this experiment argue that given a strong signal delivered through the TCR by an authentic ligand, no costimulation is required.     

ICOS-induced B7h shedding on B cells is inhibited by TLR7/8 and TLR9

We report in this study that B7h, the ligand for the ICOS costimulatory receptor, is rapidly shed from mouse B cells following either ICOS binding or BCR engagement. Shedding occurs through proteolytic cleavage that releases the extracellular ICOS-binding region of B7h. Prior exposure of B7h-expressing APCs to ICOS-expressing cells inhibits their subsequent ability to costimulate IFN-gamma and IL-4 production from CD4+ T cells. Shedding is regulated as TLR7/8 and TLR9 ligands inhibit B7h shedding. A shedding-resistant B7h mutant elicits greater costimulation of IFN-gamma production from CD4+ T cells than does wild-type B7h. These data define shedding of B7h as a novel mechanism for controlling costimulatory signaling by B7-CD28 family members that is regulated on B cells by TLR signaling.     

Characterization of endogenous Chinese hamster ovary cell surface molecules that mediate T cell costimulation

Chinese hamster ovary (CHO) cells are commonly used in the generation of transfectants for use in in vitro costimulation assays. However, we have noted that nontransfected CHO cells can themselves provide a low-level B7/CD28 independent costimulatory signal for CD3-mediated murine T cell activation and IL-2 production. This study set out to identify those molecules that contribute to this CHO-dependent costimulatory activity. We describe a CHO subline capable of delivering potent CD28-independent costimulation to murine T cells and the generation of monoclonal antibodies against these CHO cells that inhibited this costimulatory activity. These blocking antibodies do not affect CHO cell-independent costimulation or bind mouse cells, suggesting an effect mediated by their target molecules on the costimulatory competent CHO cells. Immunoprecipitation and expression cloning revealed that these antibodies bound the hamster homologues of Crry (CD21/35), CD44, CD54 (ICAM-1), CD63, CD87, CD147, and an 80- to 90-kDa protein which could not be cloned. Expression of these hamster genes on COS cells demonstrated that hamster CD54 was able to costimulate both CD3-mediated IL-2 secretion and T cell proliferation by naive murine T cells independent of the other molecules identified.     

Critical role of costimulation in the activation of naive antigen-specific TCR transgenic CD8+ T cells in vitro.

The influence of costimulation on the activation of naive CD8+ T cells and thymocytes was studied in vitro using H-Y-specific TCR-transgenic mice and H-Y antigenic peptide. Using a variety of physiological APC types, the activation of naive CD8+ T cells depended strictly on costimulation, which could not be substituted by high epitope density. T cell activation is known to be regulated by the interactions between CD86/CD80 and CD28/CD152, although it remains unclear whether the B7 isoforms have distinct roles. Addition of soluble anti-CD86 Ab led to profound inhibition of T cell reactivity, further confirming the importance of costimulation in naive CD8+ T cell activation. Finally, TCR engagement in the absence of costimulation had no effect on the subsequent reactivity of peripheral naive transgenic CD8+ T cells, but induced nonresponsiveness in mature CD8+ transgenic thymocytes. Collectively, these results demonstrate the importance of costimulation for naive CD8+ T cell activation, suggest that CD80 and CD86 can mediate opposing effects, possibly due to differential interaction with CD152 and CD28, and indicate differences in the sensitivity of immature vs mature CD8+ T cells to the induction of nonresponsiveness following costimulation-deficient Ag presentation.     

Induction of antitumor immune response by homeostatic proliferation and CD28 signaling

Inducing lymphopenia before adoptive cell transfer can improve the antitumor effect of donor immune cells. It was recently reported that lymphopenic conditions can initiate the differentiation of naive T cells into effector cells. Although T cells require a specific "strong" signal via TCR as well as costimulatory signals during Ag-driven differentiation, there has been little evidence to suggest any requirement for costimulatory signaling for the differentiation of naive T cells in a lymphopenic host. In this study, we demonstrate that naive CD8(+) T cells are indispensable for induction of antitumor effect, and, in addition to Ag-driven differentiation, CD28 signaling is essential for the differentiation of naive CD8(+) T cells into functional effector CTLs during homeostatic proliferation (HP). The systemic administration of IL-2 did not restore the antitumor effect induced by HP in the absence of CD28 signaling. These results suggest that homeostatic cytokines enable CD8(+) T cells to expand and survive, and that TCR and the CD28 signal initiate the differentiation of effector functions. A deeper understanding of the mechanisms underlying enhanced induction of the antitumor immune response with accompanying HP may allow us to more precisely induce enhanced immunity with costimulation signaling and the administration of common gamma-chain cytokines.     

Costimulation of multiple NK cell activation receptors by NKG2D

The activation of NK cells is mediated through specific interactions between activation receptors and their respective ligands. Little is known, however, about whether costimulation, which has been well characterized for T cell activation, occurs in NK cells. To study the function of NKG2D, a potential NK costimulatory receptor, we have generated two novel hamster mAbs that recognize mouse NKG2D. FACS analyses demonstrate that mouse NKG2D is expressed on all C57BL/6 IL-2-activated NK (lymphokine-activated killer (LAK)) cells, all splenic and liver NK cells, and approximately 50% of splenic NKT cells. Consistent with limited polymorphism of NKG2D, its sequence is highly conserved, and the anti-NKG2D mAbs react with NK cells from a large number of different mouse strains. In chromium release assays, we show that stimulation of NK cells with anti-NKG2D mAb can redirect lysis. Also, enhanced lysis of transfected tumor targets expressing NKG2D ligand could be inhibited by addition of anti-NKG2D mAb. Interestingly, stimulation of LAK cells via NKG2D alone does not lead to cytokine release. However, stimulation of LAK via both an NK activation receptor (e.g., CD16, NK1.1, or Ly-49D) and NKG2D leads to augmentation of cytokine release compared with stimulation through the activation receptor alone. These results demonstrate that NKG2D has the ability to costimulate multiple NK activation receptors.     

Functional Dichotomy between NKG2D and CD28-Mediated Co-Stimulation in Human CD8+ T Cells

Both CD28 and NKG2D can function as co-stimulatory receptors in human CD8⁺ T cells. However, their independent functional contributions in distinct CD8⁺ T cell subsets are not well understood. In this study, CD8⁺ T cells in human peripheral blood- and lung-derived lymphocytes were analyzed for CD28 and NKG2D expression and function. We found a higher level of CD28 expression in PBMC-derived naïve (CD45RA⁺CD27⁺) and memory (CD45RA⁻CD27⁺) CD8⁺ T cells (CD28^Hi), while its expression was significantly lower in effector (CD45RA⁺CD27⁻) CD8⁺ T cells (CD28^Lo). Irrespective of the differences in the CD28 levels, NKG2D expression was comparable in all three CD8⁺ T cell subsets. CD28 and NKG2D expressions followed similar patterns in human lung-resident GILGFVFTL/HLA-A2-pentamer positive CD8⁺ T cells. Co-stimulation of CD28^Lo effector T cells via NKG2D significantly increased IFN-γ and TNF-α levels. On the contrary, irrespective of its comparable levels, NKG2D-mediated co-stimulation failed to augment IFN-γ and TNF-α production in CD28^Hi naïve/memory T cells. Additionally, CD28-mediated co-stimulation was obligatory for IL-2 generation and thereby its production was limited only to the CD28^Hi naïve/memory subsets. MICA, a ligand for NKG2D was abundantly expressed in the tracheal epithelial cells, validating the use of NKG2D as the major co-stimulatory receptor by tissue-resident CD8⁺ effector T cells. Based on these findings, we conclude that NKG2D may provide an expanded level of co-stimulation to tissue-residing effector CD8⁺ T cells. Thus, incorporation of co-stimulation via NKG2D in addition to CD28 is essential to activate tumor or tissue-infiltrating effector CD8⁺ T cells. However, boosting a recall immune response via memory CD8⁺ T cells or vaccination to stimulate naïve CD8⁺ T cells would require CD28-mediated co-stimulation.     

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 lymphoid chemokine CCL21 costimulates naive T cell expansion and Th1 polarization of non-regulatory CD4+ T cells

CCL21 (SLC/6Ckine) is constitutively expressed by secondary lymphoid tissue and attracts CCR7-expressing mature dendritic cells and naive T cells. Recent studies demonstrated that intra-tumoral delivery of CCL21 induces tumor regression in a T cell dependent manner. CCL21 is known to mediate T cell trafficking but little is known about its function as a costimulatory molecule. Herein, we demonstrate that CCL21 costimulates expansion of CD4+ and CD8+ T cells and induces Th1 polarization. These effects were specific for naive T cells, and we show that CD4+CD25+ regulatory T cells were hyporesponsive to CCL21 induced migration, and unresponsive to CCL21 costimulation. These unique functions of CCL21 to both attract naive T cells as well as costimulate their proliferation and differentiation, suggests that CCL21 is a pivotal molecule for priming T cell responses and has therapeutic implications for local delivery of CCL21. The coordinated effects of CCL21 on T cell migration and activation may also represent a more comprehensive paradigm for the activity of other chemokines as well.     

Role of the stress kinase pathway in signaling via the T cell costimulatory receptor 4-1BB.

4-1BB is a member of the TNFR superfamily expressed on activated CD4+ and CD8+ T cells. 4-1BB can costimulate IL-2 production by resting primary T cells independently of CD28 ligation. In this study, we report signaling events following 4-1BB receptor aggregation using an Ak-restricted costimulation-dependent T cell hybridoma, C8.A3. Aggregation of 4-1BB on the surface of C8.A3 cells induces TNFR-associated factor 2 recruitment, which in turn recruits and activates apoptosis signal-regulating kinase-1, leading to downstream activation of c-Jun N-terminal/stress-activated protein kinases (JNK/SAPK). 4-1BB ligation also enhances anti-CD3-induced JNK/SAPK activation in primary T cells. Overexpression of a catalytically inactive form of apoptosis signal-regulating kinase-1 in C8.A3 T cells interferes with activation of the SAPK cascade and with IL-2 secretion, consistent with a critical role for JNK/SAPK activation in 4-1BB-dependent IL-2 production. Given the ability of both CD28 and 4-1BB to induce JNK/SAPK activation, we asked whether hyperosmotic shock, another inducer of this cascade, could function to provide a costimulatory signal to T cells. Osmotic shock of resting primary T cells in conjunction with anti-CD3 treatment was found to costimulate IL-2 production by the T cells, consistent with a pivotal role for JNK/SAPK in T cell costimulation.     

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.