Analysis of 4-1BB ligand (4-1BBL)-deficient mice and of mice lacking both 4-1BBL and CD28 reveals a role for 4-1BBL in skin allograft rejection and in the cytotoxic T cell response to influenza virus.

4-1BB ligand (4-1BBL) is a member of the TNF family expressed on activated APC. 4-1BBL binds to 4-1BB (CD137) on activated CD4 and CD8 T cells and in conjunction with strong signals through the TCR provides a CD28-independent costimulatory signal leading to high level IL-2 production by primary resting T cells. Here we report the immunological characterization of mice lacking 4-1BBL and of mice lacking both 4-1BBL and CD28. 4-1BBL-/- mice mount neutralizing IgM and IgG responses to vesicular stomatitis virus that are indistinguishable from those of wild-type mice. 4-1BBL-/- mice show unimpaired CTL responses to lymphocytic choriomeningitis virus (LCMV) and exhibit normal skin allograft rejection but have a weaker CTL response to influenza virus than wild-type mice. 4-1BBL-/-CD28-/- mice retain the CTL response to LCMV, respond poorly to influenza virus, and exhibit a delay in skin allograft rejection. In agreement with these in vivo results, allogeneic CTL responses of CD28-/- but not CD28+/+ T cells to 4-1BBL-expressing APC are substantially inhibited by soluble 4-1BB receptor as is the in vitro secondary response of CD28+ T cells to influenza virus peptides. TCR-transgenic CD28-/- LCMV glycoprotein-specific T cells are insensitive to the presence of 4-1BBL when a wild-type peptide is used, but the response to a weak agonist peptide is greatly augmented by the presence of 4-1BBL. These results further substantiate the idea that different immune responses vary in their dependence on costimulation and suggest a role for 4-1BBL in augmenting suboptimal CTL responses in vivo.     

4-1BBL induces TNF receptor-associated factor 1-dependent Bim modulation in human T cells and is a critical component in the costimulation-dependent rescue of functionally impaired HIV-specific CD8 T cells

During chronic infection, HIV-specific CD8 T cells exhibit progressive signs of functional impairment, attributed to persistent antigenic stimulation, up-regulation of the inhibitory receptor PD-1, and declining T cell help. Strategies that directly improve CD8 T cell function offer the potential of restoring immune control of HIV. Although PD-1 expression has been identified as a cause of functional impairment in HIV, in this study, PD-1 expression was observed on only a subfraction of HIV-specific CD8 T cells in a subfraction of donors, whereas HIV-specific CTL from all donors exhibited a limited repertoire of effector functions. CD137L (4-1BBL) is emerging as an important stimulator of antiviral CD8 T cell responses. Regardless of the PD-1 status of the donors, here we show that 4-1BBL, when combined with CD80 or CD70, expands a population of Ag-specific CD8 T cells expressing multiple markers of effector function, from the functionally impaired starting population. In contrast, CD70 in combination with CD80 was insufficient for these effects and the related TNF family ligand, LIGHT, had negligible activity. The unique contribution of 4-1BBL correlated with down-regulation of the proapoptotic molecule Bim in activated CD8 T cells. Decreasing the level of TNFR-associated factor 1 in T cells using small interfering RNA resulted in increased levels of Bim in the 4-1BBL-stimulated T cells. Thus, costimulation via 4-1BBL leads to TNFR-associated factor 1-dependent Bim down-modulation in T cells, resulting in increased T cell expansion. These studies identify 4-1BBL as a critical component in therapeutic strategies aimed at improving CD8 T cell function.     

Lethal granuloma disintegration in mycobacteria-infected TNFRp55-/- mice is dependent on T cells and IL-12

Genetically susceptible, TNFRp55 gene-deficient (TNFRp55-/-) mice succumb to infection with Mycobacterium avium. Before their death, M. avium-infected TNFRp55-/- mice develop granulomatous lesions that, in contrast to granulomas in wild-type syngeneic mice, undergo acute disintegration. To determine the factors involved in these events, we depleted T cell subsets or neutralized the inflammatory cytokines IFN-gamma, IL-12, or TNF in TNFRp55-/- mice infected i.v. with M. avium. Infected TNFRp55-/- mice treated with a control mAb became moribund between days 26 and 34 postinfection, showing widespread inflammatory cell apoptosis within disintegrating granulomas. In contrast, TNFRp55-/- mice depleted of either CD4+ or CD8+ cells after granuloma initiation stayed healthy until at least day 38 postinfection and showed no signs of granuloma destruction. Neutralization of IL-12, but not of IFN-gamma or TNF, also protected M. avium-infected TNFRp55-/- mice from granuloma decomposition and from premature death. Treatment with dexamethasone or with a specific inhibitor of inducible NO synthase did not prevent granuloma dissolution or death of TNFRp55-/- mice. In conclusion, granuloma disintegration in TNFRp55-/- mice is a lethal event that is dependent on IL-12 and that is mediated by an excess of T cells.     

Recombinant salmonella-based 4-1BBL vaccine enhances T cell immunity and inhibits the development of colorectal cancer in rats: in vivo effects of vaccine containing 4-1BBL

Background

Immunotherapy with vaccines is attractive for the treatment of cancer. This study is aimed at determining the effect of recombinant Salmonella (SL3261)-based 4-1BB ligand (4-1BBL) vaccine on the development of colorectal cancers and the potential immune mechanisms in rats.

Results

In comparison with that in the PBS group, similar levels of 4-1BBL expression, the frequency of T cells, IFN-γ responses, and comparable numbers of tumors were detected in the SL3261 and SL3261C groups of rats. In contrast, significantly fewer numbers of tumors, increased levels of 4-1BBL expression in the spleens and colorectal tissues, higher frequency of peripheral blood and splenic CD3⁺CD25⁺ T cells, and stronger splenic T cell IFN-γ responses were detected in the SL3261R group of rats.

Conclusion

Our results indicated that vaccination with recombinant attenuated Salmonella harboring the 4-1BBL gene efficiently enhanced T cell immunity and inhibited the development of carcinogen-induced colorectal cancers in rats.     

Innate immune activation of CD4 T cells in salmonella-infected mice is dependent on IL-18

Production of IFN-gamma by CD4 T cells is generally thought to be mediated by TCR triggering, however, Ag-nonspecific activation of effector CD8 T cells has been reported in infection models. In this study, we demonstrate that Ag-experienced CD4 T cells in the spleen of Salmonella-infected mice acquire the capacity to rapidly secrete IFN-gamma in response to stimulation with bacterial lysate or LPS. This innate responsiveness of T cells was transient and most apparent during, and immediately following, active Salmonella infection. Furthermore, innate T cell production of IFN-gamma in response to bacterial lysate or LPS was Ag independent and could be induced in Listeria-infected mice and in the absence of MHC class II expression. IL-18 was required for maximal innate responsiveness of CD4 T cells in Salmonella-infected mice and for optimal bacterial clearance in vivo. These data demonstrate that CD4 T cells acquire the capacity to respond to innate stimuli during active bacterial infection, a process that may contribute significantly to amplifying effector responses in vivo.     

4-1BB ligand, a member of the TNF family, is important for the generation of antiviral CD8 T cell responses.

4-1BB (CD137) is a costimulatory molecule expressed on activated T cells and interacts with 4-1BB ligand (4-1BBL) on APCs. To investigate the role of 4-1BB costimulation for the development of primary immune responses, 4-1BBL-deficient (4-1BBL-/-) mice were infected with lymphocytic choriomeningitis virus (LCMV). 4-1BBL-/- mice were able to generate CTL and eliminate acute LCMV infection with normal kinetics, but CD8 T cell expansion was 2- to 3-fold lower than in wild-type (+/+) mice. In the same mice, virus-specific CD4 Th and B cell responses were minimally affected, indicating that 4-1BB costimulation preferentially affects CD8 T cell responses. This result contrasts with our earlier work with CD40L-deficient (CD40L-/-) mice, in which the CD8 T cell response was unaffected and the CD4 T cell response was markedly impaired. When both 4-1BBL- and B7-dependent signals were absent, CD8 T cell expansion was further reduced, resulting in lower CTL activity and impairing their ability to clear LCMV. Altogether, these results indicate that T cells have distinct costimulatory requirements: optimal CD8 responses require 4-1BBL-dependent interactions, whereas CD4 responses are minimally affected by 4-1BB costimulation, but require CD40-CD40L and B7-dependent interactions.     

CD8+ T cell-mediated airway hyperresponsiveness and inflammation is dependent on CD4+IL-4+ T cells

CD4+ T cells, particularly Th2 cells, play a pivotal role in allergic airway inflammation. However, the requirements for interactions between CD4+ and CD8+ T cells in airway allergic inflammation have not been delineated. Sensitized and challenged OT-1 mice in which CD8+ T cells expressing the transgene for the OVA(257-264) peptide (SIINFEKL) failed to develop airway hyperresponsiveness (AHR), airway eosinophilia, Th2 cytokine elevation, or goblet cell metaplasia. OT-1 mice that received naive CD4+IL-4+ T cells but not CD4+IL-4- T cells before sensitization developed all of these responses to the same degree as wild-type mice. Moreover, recipients of CD4+IL-4+ T cells developed significant increases in the number of CD8+IL-13+ T cells in the lung, whereas sensitized OT-1 mice that received primed CD4+ T cells just before challenge failed to develop these responses. Sensitized CD8-deficient mice that received CD8+ T cells from OT-1 mice that received naive CD4+ T cells before sensitization increased AHR and eosinophil numbers in bronchoalveolar lavage fluid when challenged with allergen. In contrast, sensitized CD8-deficient mice receiving CD8+ T cells from OT-1 mice without CD4+ T cells developed reduced AHR and eosinophil numbers in bronchoalveolar lavage fluid when challenged. These data suggest that interactions between CD4+ and CD8+ T cells, in part through IL-4 during the sensitization phase, are essential to the development of CD8+IL-13+ T cell-dependent AHR and airway allergic inflammation.     

IL-6 and IL-10 induction from dendritic cells in response to Mycobacterium tuberculosis is predominantly dependent on TLR2-mediated recognition

The initial TLR-mediated interaction between Mycobacterium tuberculosis and dendritic cells is critical, since the cytokine production that ensues can greatly influence the class of adaptive immunity that is generated to the pathogen. In this study, we therefore determined the dependency on TLR2 and TLR4 for M. tuberculosis-induced cytokine production by murine dendritic cells. A key new finding of this study is that production of IL-6 and IL-10 from dendritic cells in response to M. tuberculosis is principally dependent on TLR2. The study also indicates that M. tuberculosis can induce IL-12 production in the absence of either TLR2 or TLR4, suggesting redundancy or possibly involvement of other receptors in IL-12 production. In addition, the data also reveal that lack of TLR2 or TLR4 does not impact on dendritic cell maturation or on their ability to influence the polarity of differentiating naive T cells. Collectively, data presented here provide a mechanistic insight for the contribution of TLR2 and TLR4 to tuberculosis disease progression and offer strategies for regulating IL-6 and IL-10 production in dendritic cell-based vaccine strategies.     

Athymic nude CD4+8- T cells produce IL-2 but fail to proliferate in response to mitogenic stimuli

A comparative study of immune functions of CD4+8- T cells isolated from normal and athymic nude mice by electronic cell sorting was performed. Athymic nude CD4+8- T cells expressed the TCR-associated CD3 molecule but the level of expression was significantly lower than that of normal CD4+8- T cells. Proliferative responses were studied upon stimulation by 1) the T cell mitogen Con A; 2) anti-CD3 mediated cross-linking of the CD3:TCR complex, and 3) the combined action of PMA + ionomycin. All three mitogenic stimuli caused readily detectable cell division in normal (euthymic) CD4+8- T cells. In marked contrast, none of the mitogenic stimuli induced significant proliferation in athymic nude CD4+8- T cells. The failure of athymic nude CD4+8- T cells to proliferate occurred over a wide range of mitogen concentrations and over a 4-day observation period. Neither exogenously supplied rIL-2 or mixed lymphocyte culture supernatant had any effect on the impaired proliferative response by athymic nude CD4+8- T cells. Although IL-2 was produced by athymic nude CD4+8- T cells at a reduced level when compared to normal CD4+8- T cells, it was nevertheless readily detected upon stimulation with either Con A or anti-CD3. Furthermore, stimulation of athymic nude CD4+8- T cells by anti-CD3 induced the expression of the p55 chain of IL-2R on the cell surface. Therefore, despite production of IL-2 and induced expression of IL-2R, athymic nude CD4+8- T cells failed to undergo cell division.     

IL-2/anti-IL-2 antibody complex enhances vaccine-mediated antigen-specific CD8+ T cell responses and increases the ratio of effector/memory CD8+ T cells to regulatory T cells

IL-2 is well described as a cytokine with two markedly distinct functionalities: as a necessary signal during CD4(+) and CD8(+) T cell activation/expansion and as an essential cytokine for the maintenance of CD4(+)CD25(+)FoxP3(+) T cells (regulatory T (T(REG)) cells) during homeostasis. In this study we demonstrate for the first time that, compared with the use of IL-2 alone, a complex of IL-2 and anti-IL-2 Ab (IL-2 complex) enhances the effectiveness of a viral vaccine in a mouse model with known Ag specificity. IL-2 complex led to an increase in the number of Ag-specific effector/memory CD8(+) T cells, cytokine production, and CTL lysis following Ag-specific restimulation in a vaccination setting. Our results further demonstrate that this effect is temporary and declines over the course of a few days after the IL-2 complex treatment cycle. Moreover, in contrast to the use of IL-2 alone, IL-2 complex greatly increased the ratio of effector/memory CD8(+) T cells to T(REG) cells. This phenomenon can thus potentially be used in the enhancement of immune responses to vaccination.     

IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo

We present evidence that donor-reactive CD4(+) T cells present in mice tolerant to donor alloantigens are phenotypically and functionally heterogeneous. CD4(+) T cells contained within the CD45RB(high) fraction remained capable of mediating graft rejection when transferred to donor alloantigen-grafted T cell-depleted mice. In contrast, the CD45RB(low) CD4(+) and CD25(+)CD4(+) populations failed to induce rejection, but rather, were able to inhibit rejection initiated by naive CD45RB(high) CD4(+) T cells. Analysis of the mechanism of immunoregulation transferred by CD45RB(low) CD4(+) T cells in vivo revealed that it was donor Ag specific and could be inhibited by neutralizing Abs reactive with IL-10, but not IL-4. CD45RB(low) CD4(+) T cells from tolerant mice were also immune suppressive in vitro, as coculture of these cells with naive CD45RB(high) CD4(+) T cells inhibited proliferation and Th1 cytokine production in response to donor alloantigens presented via the indirect pathway. These results demonstrate that alloantigen-specific regulatory T cells contained within the CD45RB(low) CD4(+) T cell population are responsible for the maintenance of tolerance to donor alloantigens in vivo and require IL-10 for functional activity.     

T regulatory cells 1 inhibit a Th2-specific response in vivo

We recently described a new population of CD4(+) regulatory T cells (Tr1) that inhibits proliferative responses of bystander T cells and prevents colitis induction in vivo through the secretion of IL-10. IL-10, which had been primarily described as a Th2-specific cytokine inhibiting Th1 responses, has displayed in several models a more general immune suppression on both types of effector T cell responses. Using an immediate hypersensitivity model in which BALB/c mice immunized with OVA (alum) normally generate Th2-dominated responses, we examined the ability of OVA-specific Tr1 T cell clones to inhibit OVA-specific cytokines and Ab responses. In contrast to Th2 or Th1 T cell clones, transfer of Tr1 T cell clones coincident with OVA immunization inhibited Ag-specific serum IgE responses, whereas IgG1 and IgG2a synthesis were not affected. This specific inhibition was mediated in part through IL-10 secretion as anti-IL-10 receptor Abs treatment reverted the inhibitory effect of Tr1 T cell clones. Although specifically targeted to IgE responses, Tr1 clones' inhibitory effects were more profound as they affected Ag-specific Th2 cell priming both in term of proliferative responses and cytokine secretion. These results suggest that regulatory T cells may play a fundamental role in maintaining the balance of the immune system to prevent allergic disorders.     

Differential effects of anti-IL-1R accessory protein antibodies on IL-1alpha or IL-1beta-induced production of PGE(2) and IL-6 from 3T3-L1 cells

Soluble or cell-bound IL-1 receptor accessory protein (IL-1RAcP) does not bind IL-1 but rather forms a complex with IL-1 and IL-1 receptor type I (IL-1RI) resulting in signal transduction. Synthetic peptides to various regions in the Ig-like domains of IL-1RAcP were used to produce antibodies and these antibodies were affinity-purified using the respective antigens. An anti-peptide-4 antibody which targets domain III inhibited 70% of IL-1beta-induced productions of IL-6 and PGE(2) from 3T3-L1 cells. Anti-peptide-2 or 3 also inhibited IL-1-induced IL-6 production by 30%. However, anti-peptide-1 which is directed against domain I had no effect. The antibody was more effective against IL-1beta compared to IL-1alpha. IL-1-induced IL-6 production was augmented by coincubation with PGE(2). The COX inhibitor ibuprofen blocked IL-1-induced IL-6 and PGE(2) production. These results confirm that IL-1RAcP is essential for IL-1 signaling and that increased production of IL-6 by IL-1 needs the co-induction of PGE(2). However, the effect of PGE(2) is independent of expressions of IL-1RI and IL-1RAcP. Our data suggest that domain III of IL-1RAcP may be involved in the formation or stabilization of the IL-1RI/IL-1 complex by binding to epitopes on domain III of the IL-1RI created following IL-1 binding to the IL-1RI.     

CD8 T cell-mediated killing of Cryptococcus neoformans requires granulysin and is dependent on CD4 T cells and IL-15

Granulysin is located in the acidic granules of cytotoxic T cells. Although the purified protein has antimicrobial activity against a broad spectrum of microbial pathogens, direct evidence for granulysin-mediated cytotoxicity has heretofore been lacking. Studies were performed to examine the regulation and activity of granulysin expressed by CD8 T cells using Cryptococcus neoformans, which is one of the most common opportunistic pathogens of AIDS patients. IL-15-activated CD8 T cells acquired anticryptococcal activity, which correlated with the up-regulation of granulysin. When granules containing granulysin were depleted using SrCl(2,) or when the gene was silenced using 21-nt small interfering RNA duplexes, the antifungal effect of CD8 T cells was abrogated. Concanamycin A and EGTA did not affect the antifungal effect, suggesting that the activity of granulysin was perforin independent. Following stimulation by the C. neoformans mitogen, CD8 T cells expressed granulysin and acquired antifungal activity. This activity required CD4 T cells and was dependent upon accessory cells. Furthermore, IL-15 was both necessary and sufficient for granulysin up-regulation in CD8 T cells. These observations are most consistent with a mechanism whereby C. neoformans mitogen is presented to CD4 T cells, which in turn activate accessory cells. The resultant IL-15 activates CD8 T cells to express granulysin, which is responsible for antifungal activity.     

In vivo suppression of naive CD4 T cell responses by IL-2- and antigen-stimulated T lymphocytes in the absence of APC competition

After stimulation, T cells enter a transient refractory period, promoted by IL-2, during which they are resistant to re-stimulation. We previously demonstrated that these IL-2- and Ag-stimulated refractory T cells are able to suppress the Ag-induced proliferation of naive T cells in vitro. We show here that, after adoptive transfer, these T cells are also able to suppress naive T cell proliferation in vivo. More interestingly, potently suppressive T cells can be generated directly in vivo by stimulation with Ag and supplemental IL-2. The activity of the suppressive cells is dose dependent, and the suppressor and suppressed T cells need not be restricted to the same MHC or Ag. Similar to its role in promoting T cell-mediated suppression in vitro, IL-2 is critical for the induction of suppressive activity in activated T cells in vivo. Supplemental IL-2, however, cannot overcome the suppressive activity in target T cells, indicating that suppression is not mediated by competition for this cytokine. Although the activated T cells block naive T cell proliferation, the naive cells do engage Ag and up-regulate the CD25 and CD69 activation markers after stimulation. Therefore, activated T cells stimulated in the presence of IL-2 develop MHC- and Ag-unrestricted suppressive activity. These results provide a new mechanism for competition among CD4(+) T lymphocytes, in which initial waves of responding T cells may inhibit subsequently recruited naive T cells. They further suggest a novel negative feedback loop limiting the expansion of T cell responses that may be present during vigorous immune responses or after IL-2 immunotherapy.     

IL-2, -4, and -15 differentially regulate O-glycan branching and P-selectin ligand formation in activated CD8 T cells.

The glycosyltransferase core 2 beta1-6 N-acetylglucosaminyl transferase (C2GnT1 or C2GlcNAcT1) is responsible for formation of branched structures on O-glycans present on cell surface glycoproteins. The O-glycan branch created by C2GnT1 is physiologically important insofar as only this structure can be extended and modified to yield P-selectin ligands that promote initial interactions between extravasating lymphocytes and endothelia. In mature T cells, C2GnT1 activity is thought to be induced as an intrinsic consequence of T cell activation. Through analysis of C2GnT1-dependent epitopes on CD43 and CD45RB we have found that in activated CD8(+) T cells expression of C2GnT1 was dependent upon exposure to specific cytokines rather than being induced as a direct consequence of activation. Activated CD8(+) cells became receptive to strong induction of C2GnT1 expression and P-selectin ligand expression in response to IL-2, moderate induction by IL-15, and minimal induction in response to IL-4. Our observations clarify the relationship between T cell activation and C2GnT1 expression, demonstrate the differential impact of distinct cytokines on expression of C2GnT1 activity and P-selectin ligand, and reinforce the concept that the cytokine milieu subsequent to activation can influence adhesion systems that dictate lymphocyte homing properties.     

Either IL-2 or IL-12 is sufficient to direct Th1 differentiation by nonobese diabetic T cells

Th cell differentiation from naive precursors is a tightly controlled process; the most critical differentiation factor is the action of the driving cytokine: IL-12 for Th1 development, IL-4 for Th2 development. We found that CD4(+) T cells from nonobese diabetic mice spontaneously differentiate into IFN-gamma-producing Th1 cells in response to polyclonal TCR stimulation in the absence of IL-12 and IFN-gamma. Instead, IL-2 was necessary and sufficient to direct T cell differentiation to the Th1 lineage by nonobese diabetic CD4(+) T cells. Its ability to direct Th1 differentiation of both naive and memory CD4(+) T cells was clearly uncoupled from its ability to stimulate cell division. Autocrine IL-2-driven Th1 differentiation of nonobese diabetic T cells may represent a genetic liability that favors development of IFN-gamma-producing autoreactive T cells.     

The programmed death-1 ligand 1:B7-1 pathway restrains diabetogenic effector T cells in vivo

Programmed death-1 ligand 1 (PD-L1) is a coinhibitory molecule that negatively regulates multiple tolerance checkpoints. In the NOD mouse model, PD-L1 regulates the development of diabetes. PD-L1 has two binding partners, programmed death-1 and B7-1, but the significance of the PD-L1:B7-1 interaction in regulating self-reactive T cell responses is not yet clear. To investigate this issue in NOD mice, we have compared the effects of two anti-PD-L1 Abs that have different blocking activities. Anti-PD-L1 mAb 10F.2H11 sterically and functionally blocks only PD-L1:B7-1 interactions, whereas anti-PD-L1 mAb 10F.9G2 blocks both PD-L1:B7-1 and PD-L1:programmed death-1 interactions. Both Abs had potent, yet distinct effects in accelerating diabetes in NOD mice: the single-blocker 10F.2H11 mAb was more effective at precipitating diabetes in older (13-wk-old) than in younger (6- to 7-wk-old) mice, whereas the dual-blocker 10F.9G2 mAb rapidly induced diabetes in NOD mice of both ages. Similarly, 10F.2H11 accelerated diabetes in recipients of T cells from diabetic, but not prediabetic mice, whereas 10F.9G2 was effective in both settings. Both anti-PD-L1 mAbs precipitated diabetes in adoptive transfer models of CD4(+) and CD8(+) T cell-driven diabetes. Taken together, these data demonstrate that the PD-L1:B7-1 pathway inhibits potentially pathogenic self-reactive effector CD4(+) and CD8(+) T cell responses in vivo, and suggest that the immunoinhibitory functions of this pathway may be particularly important during the later phases of diabetogenesis.