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.     

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.     

Expression of costimulatory molecules in the bovine corpus luteum

Background  

Bovine luteal parenchymal cells express class II major histocompatibility complex (MHC) molecules and stimulate class II MHC-dependent activation of T cells in vitro. The ability of a class II MHC-expressing cell type to elicit a response from T cells in vivo is also dependent on expression of costimulatory molecules by the antigen presenting cell and delivery of a costimulatory signal to the T cell. Whether bovine luteal parenchymal cells express costimulatory molecules and can deliver the costimulatory signal is currently unknown.     

TNF type 2 receptor (p75) lowers the threshold of T cell activation.

T cell activation requires a threshold amount of TCR-mediated signals, an amount that is reduced by signals mediated through costimulatory molecules expressed on the T cell surface. Here the role of TNFR2 (p75) as a putative costimulatory receptor for T cell activation was examined. It was found that p75 deficiency in CD8(+) T cells increased the requirements for TCR agonist approximately 5-fold. Furthermore, p75(-/-) T cells display a marked reduction in the proliferative response to TCR agonist. This hypoproliferative response was associated with delayed kinetics of induction of the acute activation markers CD25 and CD69 as well as a marked decrease in the production of IL-2 and IFN-gamma. The net result is that very few cells are recruited into the dividing population. Interestingly, CD28 costimulation was only partially effective in rescuing the proliferative defect of p75(-/-)CD8(+) T cells. Thus, p75 provides an important costimulatory signal in addition to that provided by CD28 toward optimal T cell proliferation.     

Intercellular adhesion molecule-2, a second counter-receptor for CD11a/CD18 (leukocyte function-associated antigen-1), provides a costimulatory signal for T-cell receptor-initiated activation of human T cells.

Activation of T cells often requires both activation signals delivered by ligation of the TCR and those resulting from costimulatory interactions between certain T cell surface accessory molecules and their respective counter-receptors on APC. CD11a/CD18 complex on T cells modulate the activation of T cells by interacting with its counter-receptors intracellular adhesion molecule (ICAM-1) (CD54) and/or ICAM-2 on the surface of APC. The costimulatory ability of ICAM-1 has been demonstrated. Using a soluble ICAM-2 Ig fusion protein (receptor globulin, Rg) we demonstrate the costimulatory effect of ICAM-2 during the activation of CD4+ T cells. When coimmobilized with anti-TCR-1 mAb ICAM-2 Rg induced vigorous proliferative response of CD4+ T cells. This costimulatory effect of ICAM-2 was dependent on its coimmobilization with mAb directed at the CD3/TCR complex but not those directed at CD2 or CD28. Both resting as well as Ag-primed CD4+ T cells responded to the costimulatory effects of ICAM-2. The addition of mAb directed at the CD11a or CD18 molecules almost completely inhibited the responses to ICAM-2 Rg. These results are consistent with the role of CD11a/CD18 complex as a receptor for ICAM-2 mediating its costimulatory effects. Stimulation of T cells with coimmobilized anti-TCR-1 and ICAM-2 resulted in the induction of IL-2R (CD25), and anti-Tac (CD25) mAb inhibited this response suggesting the contribution of endogenously synthesized IL-2 during this stimulation. These results demonstrate that like its homologue ICAM-1, ICAM-2 also exerts a strong costimulatory effect during the TCR-initiated activation of T cells. The costimulatory effects generated by the CD11a/CD18:ICAM-2 interaction may be critical during the initiation of T cell activation by ICAM-1low APC.     

A diabetogenic gene prevents T cells from receiving costimulatory signals.

T cell fate following antigen encounter is determined by several intracellular signals generated by the interaction of the T cell with an antigen-presenting cell. In the periphery activation requires T cell receptor signaling (signal one) in combination with costimulatory signals (signal two), usually provided through the cognate interaction of CD28 and B7 molecules. Provision of signal one alone to purified murine peripheral T cells in vitro induces apoptosis or anergy rather than promoting activation. These T cells can be rescued from apoptosis if they are provided with costimulation supplied, for example, by engaging the CD28 co-receptor with an anti-CD28 monoclonal antibody or by adding an exogenous source of interleukin-2. However, a majority of peripheral T cells from autoimmune, diabetes-prone Biobreeding (BB) rats exhibited different responses to these stimuli. T cells from these rats could not be rescued from apoptosis by costimulation. This was not due to the inability of BB-DP T cells to upregulate CD28 and the IL-2 receptor in response to TCR crosslinking. The failure of these costimulatory interactions to rescue BB-DP T cells segregated with the diabetes-susceptibility gene iddm1. Iddm1 in the rat causes peripheral T cell lymphopenia, which is associated with a dramatically shortened peripheral T cell life span. Our results indicate that a diabetogenic gene may contribute to autoimmunity by negating costimulatory signals important for the survival of long-lived peripheral T cells.     

Non-CD28 costimulatory molecules present in T cell rafts induce T cell costimulation by enhancing the association of TCR with rafts

While CD28 functions as the major T cell costimulatory receptor, a number of other T cell molecules have also been described to induce T cell costimulation. Here, we investigated the mechanisms by which costimulatory molecules other than CD28 contribute to T cell activation. Non-CD28 costimulatory molecules such as CD5, CD9, CD2, and CD44 were present in the detergent-insoluble glycolipid-enriched (DIG) fraction/raft of the T cell surface, which is rich in TCR signaling molecules and generates a TCR signal upon recruitment of the TCR complex. Compared with CD3 ligation, coligation of CD3 and CD5 as an example of DIG-resident costimulatory molecules led to an enhanced association of CD3 and DIG. Such a DIG redistribution markedly up-regulated TCR signaling as observed by ZAP-70/LAT activation and Ca2+ influx. Disruption of DIG structure using an agent capable of altering cholesterol organization potently diminished Ca2+ mobilization induced by the coligation of CD3 and CD5. This was associated with the inhibition of the redistribution of DIG although the association of CD3 and CD5 was not affected. Thus, the DIG-resident costimulatory molecules exert their costimulatory effects by contributing to an enhanced association of TCR/CD3 and DIG.     

Coordinate regulation of T cell activation by CD2 and CD28

T cell activation requires co-engagement of the TCR with accessory and costimulatory molecules. However, the exact mechanism of costimulatory function is unknown. Mice lacking CD2 or CD28 show only mild deficits, demonstrating that neither protein is essential for T cell activation. In this paper we have generated mice lacking both CD2 and CD28. T cells from the double-deficient mice have a profound defect in activation by soluble anti-CD3 Ab and Ag, yet remain responsive to immobilized anti-CD3. This suggests that CD2 and CD28 may function together to facilitate interactions of the T cell and APC, allowing for efficient signal transduction through the TCR.     

An intracellular calcium increase and protein kinase C activation fail to initiate T cell proliferation in the absence of a costimulatory signal

Resting T lymphocytes proliferate in response to a combination of a calcium ionophore and a phorbol ester. This observation suggests that an increase in intracellular calcium free ion concentration [Ca2+]i and activation of protein kinase C (PKC) are sufficient signaling events for the initiation of T cell proliferation. In contrast, an accessory cell-generated costimulatory signal, acting independently of the rise in [Ca2+]i and PKC activation, is required for Ag-induced proliferation of type I T cell clones. We now report that this costimulatory signal is unexpectedly also being delivered via a cell-cell interaction during the response to ionomycin and phorbol ester. In the absence of this signal (at limiting cell numbers), T cells fail to divide. We also demonstrate that proliferation in response to immobilized anti-CD3 mAb requires the cell-cell interaction. These results suggest a model of T cell stimulation in which activation of a costimulatory signaling pathway is important in the regulation of the IL-2 gene, and only in the presence of this (third) signal can an increase in [Ca2+]i and PKC activity induce T cell proliferation. Such a model predicts that IL-2-dependent expansion of T cell clones in vivo in response to Ag receptor occupancy requires the delivery of an independent accessory cell-derived co-stimulatory signal.     

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.     

The role of B7 costimulation in T-cell immunity.

CD4+ T cells are considered to be the major controlling element of the adaptive immune response. They recognize foreign peptides by interaction of the T cell receptor (TCR) with peptide complexed to major histocompatibility complex (MHC) class II molecules on the surface of antigen presenting cells (APC). Once activated, CD4+ T cells orchestrate the various phases of the immune response. They are responsible for the production of numerous cytokines, which activate specific immune effector cell populations including B cells, eosinophils, mast cells and macrophages. Not surprisingly, the activation of CD4+ T cells needs to be tightly regulated and is subject to finely tuned control mechanisms. The requirement for a second or 'costimulatory' signal, in addition to the antigenic signal, provides a key element for the exquisite control of T cell activation. One of the major signalling pathways responsible for delivery of this costimulatory signal is induced by interaction of CD28 on T cells with B7 molecules found only on APC. The present review outlines our current understanding of the physiological role of B7 costimulatory signals in regulating CD4+ T cell responses.     

Costimulatory Effect of Fas in Mouse T Lymphocytes

To induce proper immune responses, T lymphocytes require two types of stimuli, antigen-specific and costimulatory signals. Among costimulatory molecules, CD28-engagement promotes the survival and proliferation of both naive and memory T cells. In addition, it is now believed that Fas may play a role in T cell activation in the human system. It is, however, controversial whether Fas can act as a costimulatory signal in the murine system. Thus, we investigated fundamental differences in the capacity to induce proliferation of T cells between Fas and CD28 in mice. Fas-mediated T cell proliferation was observed only with a full mitogenic dose of anti-CD3 antibodies, whereas CD28 engagement was able to enhance T cell proliferation in the presence of a suboptimal level of anti-CD3 antibody. Furthermore, Fas-engaged T cells showed faster response in the upregulation of CD25 and CD69 expression than CD28-engaged ones. Here, we report that Fas might play a role in mature T cell activation in the mouse system through a different mechanism from that in CD28 costimulation.     

Costimulatory Effect of Fas in Mouse T Lymphocytes

To induce proper immune responses, T lymphocytes require two types of stimuli, antigen-specific and costimulatory signals. Among costimulatory molecules, CD28-engagement promotes the survival and proliferation of both naive and memory T cells. In addition, it is now believed that Fas may play a role in T cell activation in the human system. It is, however, controversial whether Fas can act as a costimulatory signal in the murine system. Thus, we investigated fundamental differences in the capacity to induce proliferation of T cells between Fas and CD28 in mice. Fas-mediated T cell proliferation was observed only with a full mitogenic dose of anti-CD3 antibodies, whereas CD28 engagement was able to enhance T cell proliferation in the presence of a suboptimal level of anti-CD3 antibody. Furthermore, Fas-engaged T cells showed faster response in the upregulation of CD25 and CD69 expression than CD28-engaged ones. Here, we report that Fas might play a role in mature T cell activation in the mouse system through a different mechanism from that in CD28 costimulation.     

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.     

Strength of prior stimuli determines the magnitude of secondary responsiveness in CD8+ T cells

CD8+T cells can become anergic following activation, though the cellular mechanism, as compared to CD4+ T cells, remains poorly understood. Here, we examined the effects of different antigen-dose, peptide ligands, and engagement of costimulatory molecules on the induction of CD8+ T cell anergy. We observed that increasing strengths of signals delivered to CD8+ T cells by varying the antigen-dose and the nature of peptide ligands induced increasing degrees of non-responsiveness to secondary stimulation. Furthermore, higher levels of LFA-3 engagement of CD2 rendered CD8+ T cells unresponsive to secondary antigenic re-challenge. This pattern of secondary responsiveness lasted up to 2 weeks following primary stimulation and was not correlated with prior cell division history. These results indicate that the strength of prior stimuli, which is determined by the sum of signals from both TCR and costimulatory molecules, determines the activation threshold and magnitude of CD8+ T cell responses.     

Human resting B lymphocytes can serve as accessory cells for anti-CD2-induced T cell activation.

Although resting B cells are poor accessory cells for signals transmitted through the TCR/CD3 complex, we report that these B cells can support T cell proliferation when T cell activating signals are delivered through CD2. This was first suggested when leucine methyl ester treatment of PBMC abolished proliferation induced by anti-CD3, but not by the accessory cell-dependent anti-CD2 mAb combination, GT2 and OKT11. Then we demonstrated that unstimulated, resting B cells could support the proliferation of both CD4+ and CD8+ T cells. Aggregated IgG inhibited proliferation, suggesting that anti-CD2 mAb bound to T cells were cross-linked by attachment to B cell FcR. Two lines of evidence suggested that lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interaction was crucial for anti-CD2-induced proliferation. First, proliferation was blocked by mAb against these adhesion molecules. Second, intercellular adhesion molecule-1 expression rapidly increased on resting B cells after the addition of anti-CD2, but not anti-CD3. This was of interest because fixed monocytes, but not fixed B cells, were able to support the proliferative response. In contrast to lymphocyte function-associated Ag-1/intercellular adhesion molecule-1, CD28/B7 interaction was not required for anti-CD2-induced proliferation, although ligation of these molecules provided important costimulatory signals for stimulation by anti-CD3. Finally, neutralizing antibodies against IL-1 alpha, IL-1 beta, and IL-6 showed only modest inhibitory effects on T cell proliferation. The addition of IL-1 and/or IL-6 to T cells failed to substitute for accessory cells and were only partially effective with fixed B cells. Further evidence of a linkage between CD2 and CD45 isoforms was obtained. Anti-CD45RA, but not anti-CD45RO, potentiated anti-CD2-induced T cell proliferation. These studies have revealed a novel role for resting B cells as accessory cells and have documented costimulatory signals that are important for this effect. Because Ag-presentation by resting B cells to T cells generally leads to T cell nonresponsiveness, it is possible that this tolerogenic signal may be converted to an activation signal if there is concurrent perturbation of CD2 on T cells.     

Delayed and separate costimulation in vitro supports the evidence of a transient "excited" state of CD8+ T cells during activation

Although the two-signal model for T cell activation states that a signal-1 through the TCR and a costimulatory signal-2 are required for optimal stimulation, it is now clear that the requirement for costimulation can be bypassed under certain conditions. We previously reported that this is the case for naive CD8+ T cells in vitro. In the present study we tested the effect of signal-2 when delivered after signal-1 has been disrupted. Naive CD8+ T cells from TCR transgenic mice were stimulated in vitro by using immobilized recombinant single-chain MHC molecules alone as signal-1. This signal was then stopped after different lengths of time, and anti-CD28 mAb as signal-2 was given either immediately or after a time lag. We found that signal-2 can potentiate a short signal-1 when added sequentially. Moreover, a time lag between the two signals does not abolish this potentiation. If the strength of signal-1, but not its duration, is increased, then the time lag between the delivery of signals 1 and 2 can be lengthen without loss of potentiation. Together, our results indicate that the two signals do not need to be delivered concomitantly to get optimal T cell activation. We suggest that the CD8+ T cells can reach a transient "excited" state after being stimulated with signal-1 alone, characterized by the cell's ability to respond to separate and delayed signal-2.     

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.     

CD28 delivers a costimulatory signal involved in antigen-specific IL-2 production by human T cells.

CD4+ T cells require two signals to produce maximal amounts of IL-2, i.e., TCR occupancy and an unidentified APC-derived costimulus. Here we show that this costimulatory signal can be delivered by the T cell molecule CD28. An agonistic anti-CD28 mAb, but not IL-1 and/or IL-6, stimulated T cell proliferation by tetanus toxoid-specific T cells cultured with Ag-pulsed, costimulation-deficient APC. Furthermore, the ability of B cell tumor lines to provide costimulatory signals to purified T cells correlated well with expression of the CD28 ligand B7/BB-1. Finally, like anti-CD28 mAb, autologous human APC appeared to stimulate a cyclosporine A-resistant pathway of T cell activation. Together, these results suggest that the two signals required for IL-2 production by CD4+ T cells can be transduced by the TCR and CD28.