Robert J. Rossi

Effector CD8 T cells possess suppressor function after 4-1BB and Toll-like receptor triggering

To better understand how innate and adaptive immune responses interact with each other, we combined 4-1BB T cell costimulation with specific adjuvants to determine whether these treatments would influence specific T cell expansion and function in vivo. In the presence of 4-1BB ligation and Toll-like receptor 3 (TLR)3 and/or TLR4 triggering, CD8 T cell clonal expansion and survival was augmented profoundly. Specific T cells primed in vivo with TLR ligands responded normally to in vitro recall stimulus, but, surprisingly, copriming with 4-1BB costimulation significantly impaired the recall response even though many more specific effector T cells were rescued in vivo. Here, we demonstrate that the rescued CD8 T cells suppressed CD4 T cell proliferation via a type β transforming growth factor-dependent mechanism. Thus, 4-1BB and TLR ligands induce survival of specific effector CD8 T cells with suppressive recall potential, which may explain the dual role that 4-1BB activation plays in mediating tumor clearance and prevention of autoimmune disease.

IL-18 Bridges Innate and Adaptive Immunity through IFN-γ and the CD134 Pathway

IL-18 induces inflammation resulting in either enhanced protection from pathogens or exacerbation of autoimmunity, and T cells are profoundly activated during these responses. How IL-18 influences T cell activation is unknown, but this study in mice shows that IL-18 boosted Ag-specific T cell clonal expansion of effector T cells and induced a subpopulation of IFN-γ superproducing T cells. Commitment to IFN-γ production through IL-18 was independent of NK cells and IL-12 but dependent on host-derived IFN-γ. To determine how expansion of these effectors occurred, IL-18 was shown to induce OX40L on dendritic cells, whereas peptide stimulation induced CD134 (OX40) on specific T cells. CD134 blockade inhibited T cell effector expansion thereby reducing the number of IFN-γ superproducers by 12-fold. Thus, independent of IL-12, IL-18 impacts T cell immunity throughout lymphoid and nonlymphoid tissue by bridging the innate and adaptive arms of the immune system through IFN-γ and the CD134 costimulatory pathway.

T Cell Clonal Conditioning: A Phase Occurring Early after Antigen Presentation but before Clonal Expansion Is Impacted by Toll-Like Receptor Stimulation

After in vivo immunization, Ag-specific T cells disappear from circulation and become sequestered in lymphoid tissue where they encounter Ag presented by dendritic cells. In the same site and just after Ag presentation, they “disappear” a second time and we investigated this process. Using a mouse model of T cell deletion (without Toll-like receptor (TLR) stimulation) vs survival (with TLR stimulation), Ag-specific T cells indeed became undetectable by flow cytometry, however were readily detected by immunohistochemistry. Thus, whether or not the activated T cells were destined to delete or survive, they were difficult to extract from lymphoid tissue and did not disappear but in fact were abundantly present. Nevertheless, profound differences were observed during this time period when tolerizing conditions were compared with immunizing conditions. TLR stimulation induced an increase in CD25 expression, acquisition of surface MHC class II, and abnormally high increases in forward and side scatter of the peptide-specific T cells. Using a modified adoptive transfer approach, we demonstrated by flow cytometry that in the presence of TLR stimulation the Ag-specific T cells were tightly coupled to dendritic cells, explaining the unusual increases in size and granularity. Ultimately, these events induced the specific T cells to differentiate into memory cells. We postulate that this is a stage where T cells are either conditioned to survive or to delete depending upon the activation status of the innate immune system.

CD134 Costimulation Couples the CD137 Pathway to Induce Production of Supereffector CD8 T Cells That Become IL-7 Dependent

The TNFR superfamily members 4-1BB (CD137) and OX40 (CD134) are costimulatory molecules that potently boost CD8 and CD4 T cell responses. Concomitant therapeutic administration of agonist anti-CD137 and -CD134 mAbs mediates rejection of established tumors and fosters powerful CD8 T cell responses. To reveal the mechanism, the role of CD137 expression by specific CD8 T cells was determined to be essential for optimal clonal expansion and accumulation of effector cells. Nonetheless, dual costimulation induced production of supereffector CD8 T cells when either the specific T cells or the host alone bore CD137. Perhaps surprisingly, the total absence of CD137 prevented anti-CD134 augmentation of supereffector differentiation demonstrating an unappreciated link between these related pathways. Ultimately, it was reasoned that these powerful dual costimulatory responses involved common γ family members, and we show substantial increases of CD25 and IL-7Rα-chain expression by the specific CD8 T cells. To investigate this further, it was shown that IL-7 mediated T cell accumulation, but importantly, a gradual and preferential effect of survival was directed toward supereffector CD8 T cells. In fact, a clear enhancement of effector differentiation was demonstrated to be proportional to the increasing amount of IL-7Rα expression by the specific CD8 T cells. Therefore, dual costimulation through CD137 and CD134 drives production and survival of supereffector CD8 T cells through a distinct IL-7-dependent pathway.

The Lipopolysaccharide Adjuvant Effect on T Cells Relies on Nonoverlapping Contributions from the MyD88 Pathway and CD11c+ Cells

Bacterial LPS is a natural adjuvant that induces profound effects on T cell clonal expansion, effector differentiation, and long-term T cell survival. In this study, we delineate the in vivo mechanism of LPS action by pinpointing a role for MyD88 and CD11c+ cells. LPS induced long-term survival of superantigen-stimulated CD4 and CD8 T cells in a MyD88-dependent manner. By tracing peptide-stimulated CD4 T cells after adoptive transfer, we showed that for LPS to mediate T cell survival, the recipient mice were required to express MyD88. Even when peptide-specific CD4 T cell clonal expansion was dramatically boosted by enforced OX40 costimulation, OX40 only synergized with LPS to induce survival when the recipient mice expressed MyD88. Nevertheless, these activated, but moribund, T cells in the MyD88−/− mice acquired effector properties, such as the ability to synthesize IFN-γ, demonstrating that effector differentiation is not automatically coupled to a survival program. We confirmed this notion in reverse fashion by showing that effector differentiation was not required for the induction of T cell survival. Hence, depletion of CD11c+ cells did not affect LPS-driven specific T cell survival, but CD11c+ cells were paramount for optimal effector T cell differentiation as measured by IFN-γ potential. Thus, LPS adjuvanticity is based on MyD88 promoting T cell survival, while CD11c+ cells support effector T cell differentiation.

Inhalation of Staphylococcus aureus Enterotoxin A Induces IFN-γ and CD8 T Cell-Dependent Airway and Interstitial Lung Pathology in Mice

Staphylococcus aureus, a primary source of bacterial superantigen (SAg), is known to colonize the human respiratory tract and has been implicated in airway inflammation. Studies have documented a role for SAgs in respiratory disorders, such as nasal polyps, chronic obstructive pulmonary disease, chronic rhinosinusitis, and asthma. However, cellular and molecular mediators involved in SAg-mediated pulmonary disease have not been clearly identified. In this study, we investigated the effect of intranasal staphylococcal enterotoxin A (SEA) exposure on murine lung. The pathological features in the lung resulting from SEA exposure had characteristics of both obstructive and restrictive pulmonary disorders. There was also an increase in bronchoalveolar lavage protein concentration and cellularity following SEA challenge. Massive CD8+Vβ3+ T cell accumulation observed in the lung was dependent on CD4 T cell help, both for recruitment and for IFN-γ synthesis. The primary source of IFN-γ synthesis was CD8 T cells, and depletion of these cells abrogated disease. IFN-γ deficiency also prevented SEA-mediated disease, and this was by enhancing early recruitment of neutrophils as detected in the bronchoalveolar lavage. Thus, IFN-γ appeared to selectively aid the recruitment of T cells to the lungs while preventing the neutrophil accumulation. Therefore, our results show that IFN-γ-producing CD8 T cells mediated pulmonary alveolitis and inflammation, which were dependent upon CD4 T cells for their recruitment to the lung.

Lipopolysaccharide Potentiates Effector T Cell Accumulation into Nonlymphoid Tissues through TRIF

LPS is a natural adjuvant that potentiates Ag-specific T cell survival and Th1 differentiation by stimulating MyD88 and Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF) signaling pathways. In this study, we reveal the TRIF pathway is critical for amplifying murine effector T cell accumulation into nonlymphoid tissues following immunization with Ag plus LPS. Although LPS increased the accumulation of splenic T cells in TRIF-deficient mice, markedly fewer T cells were recovered from liver and lung in comparison to wild type. Most of the T cells primed in TRIF-deficient mice failed to up-regulate CXCR3 and had an overall reduced capacity to produce IFN-γ, demonstrating effector T cell differentiation was linked to their migration. To investigate the role of TRIF-dependent cytokines, neutralization studies were performed in wild type mice. Although TNF neutralization reduced T cell numbers, its coneutralization with IL-10 unexpectedly restored the T cells, suggesting the balance between pro- and anti-inflammatory cytokines influences T cell survival rather than their magnitude. To investigate a role for costimulatory molecules, we tested whether the T cell defect in TRIF-deficient mice could be corrected with enforced costimulation. Boosting with a CD40 agonist in addition to LPS restored the effector CD8 T cell response in livers of TRIF-deficient mice while only partially restoring CD4 T cells, suggesting that LPS primes CD8 and CD4 T cell immunity through different mechanisms. Overall, our data support targeting TRIF for vaccines aimed to direct immune responses to nonlymphoid tissues.

TGFβ Protein Processing and Activity through TCR Triggering of Primary CD8+ T Regulatory Cells

In general, TGFβ is synthesized as a procytokine that requires proteolytic activation, release of the mature cytokine from its noncovalently associated latent-associated peptide, and binding to TGFβRII to mediate suppressive activity. We tracked this process in mice containing primed CD8 regulatory T cells (Tregs) by immunoblotting in primary whole cell lysates for pro-TGFβ, latent-associated peptide and mature TGFβ. Generation of CD8 Tregs promoted processing of the 50 kDa pro-TGFβ protein into a 12.5 kDa mature TGFβ species in vivo. Despite the inability to detect mature TGFβ in the sera of mice with primed CD8 Tregs and in the synthetic culture medium of stimulated CD8 Tregs, we demonstrated engagement of TGFβRII through immunoblotting for Smad2 phosphorylation. This process relied on continual TCR triggering, which also induced Smad3 phosphorylation. To understand the movement of mature TGFβ, we showed that in contrast to IFN-γ, mature TGFβ does not remain a soluble cytokine but is likely to be rapidly adsorbed by neighboring cells. These data show the exquisite local control directed toward TGFβ by the immune system and underscore the fine specificity involved in its detection.

The Oxazolidinone Derivative Locostatin Induces Cytokine Appeasement

Damaging inflammation arising from autoimmune pathology and septic responses results in severe cases of disease. In both instances, anti-inflammatory compounds are used to limit the excessive or deregulated cytokine responses. We used a model of robust T cell stimulation to identify new proteins involved in triggering a cytokine storm. A comparative proteomic mining approach revealed the differential mapping of Raf kinase inhibitory protein after T cell recall in vivo. Treatment with locostatin, an Raf kinase inhibitory protein inhibitor, induced T cell anergy by blocking cytokine production after Ag recall. This was associated with a reduction in Erk phosphorylation. Importantly, in vivo treatment with locostatin profoundly inhibited TNF-α production upon triggering the Ag-specific T cells. This effect was not limited to a murine model because locostatin efficiently inhibited cytokine secretion by human lymphocytes. Therefore, locostatin should be a useful therapeutic to control inflammation, sepsis, and autoimmune diseases.

Recruitment and in situ Renewal Regulate Rapid Accumulation of CD11c+ Cells in the Lung following Intranasal Superantigen Challenge

Background: Staphylococcusaureus, a primary source of bacterial superantigen, is known to colonize the human respiratory tract and has been implicated in airway inflammation. The potential pathological effect of staphylococcal enterotoxins on the respiratory tract necessitates a detailed understanding of how they regulate innate immune cells, particularly CD11c-expressing dendritic cells (DCs). Methods: C57BL/6 mice were challenged intranasally with staphylococcal enterotoxin A (SEA) and at indicated time points lung tissue was perfused, digested and analyzed for CD11c+ expressing cells. Results: The pulmonary CD11c+ cells can be divided into two major populations based on their MHC II expression. One day following intranasal SEA challenge, there was rapid accumulation of CD11c+ cells expressing medium to high levels of MHC II. The peak accumulation of CD11c+ MHC II– population was observed 2 days after SEA challenge; however, careful examination of this cell population revealed that they were heterogeneous, being comprised of cells bearing CD3, CD19, NK1.1 and F4/80 along with varying levels of CD11c. Nevertheless, there was a 2-fold increase of CD11c+ MHC II– (CD3– CD19– NK1.1– F4/80–) cells in the lungs. Conclusion: The mechanism of increase in the CD11c+ MHC II– immune progenitor population was mainly due to cellular division rather than migration from blood to lung. In contrast, the early and rapid accumulation of CD11c+ MHC IIhi cells, conventionally known as DCs, in the lung on day 1 was mostly due to migration from blood. Thus this study examines the pulmonary innate immune response to a powerful immune stimulus.