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.     

Tumor-specific CD4+ T cells are activated by "cross-dressed" dendritic cells presenting peptide-MHC class II complexes acquired from cell-based cancer vaccines

Tumor cells that constitutively express MHC class I molecules and are genetically modified to express MHC class II (MHC II) and costimulatory molecules are immunogenic and have therapeutic efficacy against established primary and metastatic cancers in syngeneic mice and activate tumor-specific human CD4+ T lymphocytes. Previous studies have indicated that these MHC II vaccines enhance immunity by directly activating tumor-specific CD4+ T cells during the immunization process. Because dendritic cells (DCs) are considered to be the most efficient APCs, we have now examined the role of DCs in CD4+ T cell activation by the MHC II vaccines. Surprisingly, we find that DCs are essential for MHC II vaccine immunogenicity; however, they mediate their effect through "cross-dressing." Cross-dressing, or peptide-MHC (pMHC) transfer, involves the generation of pMHC complexes within the vaccine cells, and their subsequent transfer to DCs, which then present the intact, unprocessed complexes to CD4+ T lymphocytes. The net result is that DCs are the functional APCs; however, the immunogenic pMHC complexes are generated by the tumor cells. Because MHC II vaccine cells do not express the MHC II accessory molecules invariant chain and DM, they are likely to load additional tumor Ag epitopes onto MHC II molecules and therefore activate a different repertoire of T cells than DCs. These data further the concept that transfer of cellular material to DCs is important in Ag presentation, and they have direct implications for the design of cancer vaccines.     

CD8+ CD28- T regulatory lymphocytes inhibiting T cell proliferative and cytotoxic functions infiltrate human cancers

Tumor growth is allowed by its ability to escape immune system surveillance. An important role in determining tumor evasion from immune control might be played by tumor-infiltrating regulatory lymphocytes. This study was aimed at characterizing phenotype and function of CD8+ CD28- T regulatory cells infiltrating human cancer. Lymphocytes infiltrating primitive tumor lesion and/or satellite lymph node from a series of 42 human cancers were phenotypically studied and functionally analyzed by suppressor assays. The unprecedented observation was made that CD8+ CD28- T regulatory lymphocytes are almost constantly present and functional in human tumors, being able to inhibit both T cell proliferation and cytotoxicity. CD4+ CD25+ T regulatory lymphocytes associate with CD8+ CD28- T regulatory cells so that the immunosuppressive activity of tumor-infiltrating regulatory T cell subsets, altogether considered, may become predominant. The infiltration of regulatory T cells seems tumor related, being present in metastatic but not in metastasis-free satellite lymph nodes; it likely depends on both in situ generation (via cytokine production) and recruitment from the periphery (via chemokine secretion). Collectively, these results have pathogenic relevance and implication for immunotherapy of cancer.     

CD28, TNF receptor,and IL-12 are critical for CD4-independent cross-priming of therapeutic antitumor CD8+ T cells

Previously, we have shown that priming of therapeutic CD8(+) T cells in tumor vaccine-draining lymph nodes of mice vaccinated with GM-CSF secreting B16BL6 melanoma cells occurs independent of CD4 T cell help. In this study, we examined the contribution of the major costimulatory molecules, CD40 ligand (CD40L), CD80, and CD86, in the priming of CD8(+) T cells. Priming of therapeutic CD8(+) T cells by a GM-CSF-transduced tumor vaccine did not require CD40 and CD40L interactions, as therapeutic T cells could be generated from mice injected with anti-CD40L Ab and from CD40L knockout mice. However, costimulation via either CD80 or CD86 was required, as therapeutic T cells could be generated from mice injected with either anti-CD80 or anti-CD86 Ab alone, but administration of both Abs completely inhibited the priming of therapeutic T cells. Blocking experiments also identified that priming of therapeutic T cells in MHC class II-deficient mice required TNFR and IL-12 signaling, but signaling through CD40, lymphotoxin-betaR, or receptor activator of NF-kappaB was not essential. Thus, cross-priming of therapeutic CD8(+) T cells by a tumor vaccine transduced with GM-CSF requires TNFR, IL-12, and CD28 signaling.     

Induction of polyclonal CD8+ T cell activation and effector function by Pertussis toxin

Pertussis toxin (PTX) has pronounced adjuvant activity and strongly enhances innate and adaptive immune responses, including increased antibody production and Th1/Th2 cytokine production. Adjuvant effects of PTX on Th1 and Th2 cells are primarily mediated via CD80/86 costimulation via enhanced expression of these molecules by APCs. However, it has remained unresolved whether PTX modulates the expression of costimulatory and inhibitory molecules on CD4+ and CD8+ T cells. To address this question, we determined the expression kinetics of CD28, CTLA-4, and CD40L on spleen CD4+ and CD8+ T cells after incubation with PTX. The results show that PTX upregulated the expression of CD28 by CD8+ T cells, but not by CD4+ T cells. In contrast, the expression of CTLA-4 and CD40L was not substantially altered on CD4+ or CD8+ T cells. CD28 upregulation by CD8+ T cells was paralleled by upregulation of CD69 and the induction of IFN-γ, Granzyme B (GrB), and IL-17. CD8+ T cell activation and cytokine production could be substantially blocked with anti-CD80 and CD86 antibodies, consistent with CD28 mediated signaling. Treatment of highly purified CD8+ T cells with PTX resulted in upregulation of CD28 and CD69, and production of IFN-γ. Incubation with CD28 mAb further enhanced this effect, suggesting that PTX has direct effects on CD8+ T cells which are enhanced by CD80/86-mediated costimulation provided by APCs.     

Lipopolysaccharide stimulates the proliferation of human CD56+CD3- NK cells: a regulatory role of monocytes and IL-10

NK cells recognize and kill tumor cells and normal cells, and these play an important role in immune defense in cancer, infectious disease, and autoimmunity. NK killing is regulated by positive or negative signals derived from the interaction of surface receptors with ligands on the target cells. However, the mechanisms controlling the proliferation and maintenance of NK cells in normal human individuals are less clearly defined. In this study, using an entirely autologous system, we demonstrate that human peripheral blood CD3-CD56+, killer cell-inhibitory receptor (KIR)-expressing cells proliferate and expand in response to LPS. These responses are enhanced in the presence of anti-IL-10 receptor-blocking Abs or on the removal of CD14+ cells from the cultures. This enhancement is also reflected in substantial increases in cytolytic activity and IFN-gamma production. The negative effect of CD14+ cells may also be IL-10 mediated, IL-10 being lost from the culture supernatants of CD14-depleted PBMC and rIL-10 reversing the effect of this depletion. On the other hand, mRNA for the p35 and p40 subunits of IL-12 is still induced in CD14-depleted cultures. The expansion of CD3-CD56+ cells was also inhibited by CTLA4-Ig, indicating a role for CD80/86. B lymphocytes were not required for the expansion of CD3-CD56+ cells, whereas removal of MHC class II+ cells from CD14-depleted cultures resulted in a complete abrogation of these responses. Expansion of CD3-CD56+ cells was reconstituted in MHC class II-depleted cell cultures by adding back monocyte-derived dendritic cells. These results indicate that the responses of CD3-CD56+ NK cells to LPS may be driven by a MHC class II+ B7+ CD14- peripheral population, most likely blood dendritic cells.     

Ligation of CD80 is critical for high-level CD25 expression on CD8+ T lymphocytes

CD80 and CD86 have been shown to play a critical role in the optimal activation of T cells. Although these two molecules bind the same ligand, CD28, the question of whether CD80 and CD86 provide unique signals or serve redundant roles remains controversial. Previous studies have suggested that CD80 binding to CD28 may be superior to CD86 for the activation of naive CD8+ T cells. This study provides a potential mechanism to explain these observations. Our study demonstrates a previously unappreciated role for CD80, its superiority over CD86 in promoting CD25 expression, increasing both the number of cells that express CD25 and the level expressed on a per cell basis. These findings provide new insights into the role of CD80 vs CD86 and have important implications for the design of vaccines and immunotherapeutics aimed at the generation of a robust CD8+ T cell response in vivo.     

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 and CD86 control antiviral CD8+ T-cell function and immune surveillance of murine gammaherpesvirus 68

The interactions between CD80 and CD86 on antigen-presenting cells and CD28 on T cells serve as an important costimulatory signal in the activation of T cells. Although the simplistic two-signal hypothesis has been challenged in recent years by the identification of different costimulators, this classical pathway has been shown to significantly impact antiviral humoral and cellular immune responses. How the CD80/CD86-CD28 pathway affects the control of chronic or latent infections has been less well characterized. In this study, we investigated its role in antiviral immune responses against murine gammaherpesvirus 68 (MHV-68) and immune surveillance using CD80/CD86(-/-) mice. In the absence of CD80/CD86, primary antiviral CD8(+) T-cell responses and the induction of neutralizing antibodies were severely impaired. During long-term immune surveillance, the virus-specific CD8(+) T cells were impaired in IFN-gamma production and secondary expansion and exhibited an altered phenotype. Surprisingly, a low level of viral reactivation in the lung was observed, and this effect was independent of CD28 and CTLA-4. Thus, CD80 and CD86, signaling through CD28 and possibly another unidentified receptor, are required for optimal immune surveillance and antiviral immune responses to murine gammaherpesvirus.     

Emergence of regulatory CD4+ T cell response to repetitive stimulation with antigen-presenting cells in vitro: implications in designing antigen-presenting cell-based tumor vaccines

Because APCs play a crucial role in the generation of T cell-mediated immune responses, numerous clinical trials with APC-based vaccines have been initiated in different types of human cancers. Encouraging results have emerged from some of these initial studies. Thus far, APC-based vaccinations usually include multiple rounds of immunization. With this approach, although we and others have detected induction of Ag-specific CTL responses in vaccinated patients after stimulation with the same APC-based immunogen, in vitro we also find that repetitive in vitro stimulation with Ag-loaded APC can, at times, lead to the emergence of noncytolytic CD4+ T cells exhibiting the characteristic phenotype of Th2 cells. These noncytolytic CD4+ T cells synthesize large quantities of type 2 cytokines such as IL-4 and IL-10 on stimulation with the autologous APC or tumor cells in an MHC class II-restricted manner. Further, these CD4+ T cells and a cell-free supernatant factor block the activation of fresh T lymphocytes. The supernatant factor also exhibits a marked inhibitory effect on the expression of the costimulatory molecules, CD80 and CD86, by APC. The inhibitory effect of the supernatant factor can be abrogated by neutralizing IL-10 in the supernatant. These observations therefore have implications in the APC-based tumor vaccine protocol design.     

Induction and large-scale expansion of CD8+ tumor specific cytotoxic T lymphocytes from peripheral blood lymphocytes by in vitro stimulation with CD80-transfected autologous melanoma cells.

Human melanoma cell lines may induce a specific T cell response against tumor cells in vitro. However, after repeated restimulation with autologous tumor cells, expansion of CTL is limited and often apoptosis of the T cells occurs. In order to improve conditions inducing primary T cell responses and thus allowing further expansion of tumor specific T cells for an adoptive transfer, we transfected human melanoma cells with the B7.1 gene (CD80), known to be a potent costimulatory molecule for T cell activation. CD80 expression on melanoma cells resulted in improved primary T cell activation, especially of CD8+ T cells. Furthermore, restimulation with CD80+ tumor cells gave rise to long term proliferating CD8+ T cell lines demonstrating an 100-fold expansion of T cells compared to the 20-30-fold increased numbers obtained with the controls (parental tumor cells +/- anti-CD28). T cells stimulated with CD80+ melanoma cells were found to display a MHC class I-restricted cytotoxic activity against the autologous tumor cells. In conclusion, these studies demonstrate the requirement of costimulation in generating large numbers of tumor specific T cells in vitro that may be used for an adoptive transfer in tumor immunotherapy.     

Tyrosine phosphorylation of a novel 100-kDa protein coupled to CD28 in resting human T cells is enhanced by a signal through TCR/CD3 complex

For T cell activation, two signals are required, i.e., a T cell receptor (TCR)/CD3-mediated main signal and a CD28-mediated costimulatory signal. CD28 binds to its ligand (CD80 or CD86) and transduces the most important costimulatory signal. The cytoplasmic domain of the CD28 molecule, composed of 41 amino acids, does not contain any intrinsic enzyme activity. The cytoplasmic domain of CD28 is remarkably conserved among species and is associated with a number of signaling molecules that affect the main signal. We report here that a tyrosine phosphorylated 100-kDa protein (ppl00) was coupled to the CD28 cytoplasmic domain in Jurkat and human peripheral T cells. The pp100 was distinguished from other CD28 associated molecules such as Vav, STAT5, PI 3-kinase, Valosin-containing protein (VCP), Nucleolin, Gab2 (Grb2-associated binding protein 2), and STAT6. The tyrosine phosphorylation of pp100 coprecipitated with CD28 was enhanced by CD3 stimulation by the specific antibody, tyrosine phosphatase inhibitor and PKC activator. Tyrosine phosphorylation of pp100 was attenuated by the prior addition of PKC inhibitor. These findings indicate that pp100 is a novel tyrosine phosphorylated protein coupled to CD28 under continuous control of tyrosine phosphatases and might play a role in T cell activation augmented by a TCR/CD3-mediated main signal.     

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.     

Porcine CTLA4-Ig lacks a MYPPPY motif, binds inefficiently to human B7 and specifically suppresses human CD4+ T cell responses costimulated by pig but not human B7

The CTLA4 receptor (CD152) on activated T lymphocytes binds B7 molecules (CD80 and CD86) on APC and delivers a signal that inhibits T cell proliferation. Several regions involved in binding to B7 are known, but the relative importance of these is not clear. We have cloned porcine CTLA4 (pCTLA4). Although highly homologous to human CTLA4 (hCTLA4), the predicted protein sequence contains a leucine for methionine substitution at position 97 in the MYPPPY sequence. A fusion protein constructed from the extracellular regions of pCTLA4 and the constant regions of human IgG1 (pCTLA4-Ig) bound porcine CD86 with equivalent affinity to that of hCTLA4-Ig. However, pCTLA4-Ig bound poorly to human CD80 and CD86 expressed on transfectants and EBV-transformed human B cells. In functional assays with MHC class II-expressing porcine endothelial cells and human B cells, pCTLA4-Ig blocked human CD4+ T cell responses to pig but not human cells, whereas control hCTLA4-Ig inhibited responses to both. Comparison between mouse, human, and porcine CTLA4-Ig suggests that the selective binding of pCTLA4-Ig to porcine CD86 molecules is due to the L for M substitution at position 97. Our results indicate that pCTLA4-Ig may be a useful reagent to define the precise nature of the interaction between B7 and CTLA4. By failing to inhibit the delivery of costimulatory signals provided by human B7, it may also prove to be a relatively specific inhibitor of the direct human T cell response to immunogenic pig tissue.     

Human epidermal cells from ultraviolet light-exposed skin preferentially activate autoreactive CD4+2H4+ suppressor-inducer lymphocytes and CD8+ suppressor/cytotoxic lymphocytes

In vivo exposure of human epidermis to UV abrogates the function of T6+DR+ Langerhans cells and induces the appearance of Ag-presenting T6-DR+ OKM5+ cells in the epidermis. Since UV exposure of murine skin results in Ts lymphocyte activation, we investigated the capacity of human epidermal cells (EC) harvested 3 days after in vivo UV exposure to activate regulatory and effector autologous T lymphocyte subsets. T lymphocytes were separated into CD8+ suppressor/cytotoxic lymphocytes and CD4+ helper/inducer lymphocytes by C lysis and panning. The CD4+ subset was further divided by using the 2H4 mAB to obtain CD4+2H4+ lymphocytes (inducers of TS lymphocytes) and CD4+2H4- lymphocytes (inducers of B cell Ig production and inducers of cytotoxic T cells). Unirradiated suction blister-derived EC from control skin (C-EC) and from skin exposed in vivo to UV (UV-EC) were cultured with purified autologous T lymphocyte subsets in the absence of added Ag. The resultant T lymphocyte proliferation was detected by [3H]thymidine uptake. UV-EC were highly effective in the stimulation of CD4+ lymphocytes, whereas C-EC were poor stimulators. The stimulator effect of UV-EC was abrogated after depletion of DR+ UV-EC. When CD4+ lymphocytes were fractionated, UV-EC consistently demonstrated enhanced ability to stimulate suppressor-inducer CD4+2H4+ lymphocytes relative to C-EC. Although less responsive than CD4+2H4+ lymphocytes, CD4+2H4- lymphocytes also demonstrated greater proliferation to UV-EC than to C-EC. Neither UV-EC nor C-EC were able to activate CD8+ lymphocytes devoid of CD4+ lymphocytes. However, after addition of rIL-2 at concentrations that allow binding only to the high affinity IL-2R on T lymphocytes, UV-EC induced vigorous proliferation of CD8+ lymphocytes, whereas C-EC induced only background levels of proliferation. C lysis of leukocytes resident within UV-EC resulted in 66 to 70% reduction of CD8+ lymphocyte proliferation. In conclusion, UV-EC may activate CD8+ lymphocytes by at least two pathways: (1) UV-EC activation of CD4+2H4+ lymphocytes may induce differentiation/proliferation of CD8+ suppressor cells and (2) UV-EC activation of CD4+ cells may induce IL-2 production, that, in combination with UV-induced epidermal leukocytes, stimulates CD8+ 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.     

Activation requirements for CD4+ T cells differing in CD45R expression.

Murine CD4+ T cells can be subdivided into naive and memory T cells based on surface phenotype, on recall response to Ag, and on differences in activation requirements. Furthermore, several studies have shown that two signals are required for CD4+ T cell activation; one signal is provided by occupancy of the TCR and the other signal is provided by the APC. In this report, analysis of naive and memory CD4 T cells, separated on the basis of CD45 isoform expression, has shown that their requirements for two signals differ. Activation of memory CD4 T cells to proliferate and secrete IL-2/IL-4 only required occupancy of the TCR complex, whereas activation of naive CD4 T cells required an APC-derived signal as well. Moreover, the signal induced by anti-CD3 antibodies differs from the signal provided by anti-V beta cross-linking of the TCR because both antibodies activate memory CD4 T cells but only anti-CD3 activates naive CD4 T cells. Together these data suggest that the consequence of stimulation through the TCR/CD3 signal complex differs between memory and naive CD4 T cells.     

Minor structural changes in a mutated human melanoma antigen correspond to dramatically enhanced stimulation of a CD4+ tumor-infiltrating lymphocyte line

While most immunotherapies for cancer have focused on eliciting specific CD8+ cytotoxic T lymphocyte killing of tumor cells, a mounting body of evidence suggests that stimulation of anti-tumor CD4+ T cell help may be required for highly effective therapy. Several MHC class II-restricted tumor antigens that specifically activate such CD4+ helper T lymphocytes have now been identified, including one from a melanoma tumor that is caused by a single base-pair mutation in the glycolytic enzyme triosephosphate isomerase. This mutation results in the conversion of a threonine residue to isoleucine within the antigenic epitope, concomitant with a greater than five log-fold increase in stimulation of a CD4+ tumor-infiltrating lymphocyte line. Here, we present the crystal structures of HLA-DR1 in complex with both wild-type and mutant TPI peptide antigens, the first structures of tumor peptide antigen/MHC class II complexes recognized by CD4+ T cells to be reported. These structures show that very minor changes in the binding surface for T cell receptor correspond to the dramatic differences in T cell stimulation. Defining the structural basis by which CD4+ T cell help is invoked in an anti-tumor immune response will likely aid the design of more effective cancer immunotherapies.