Rajshekhar Alli

IL-10 modulates DSS-induced colitis through a macrophage – ROS – NO axis

Breakdown of the epithelial barrier because of toxins or other insults leads to severe colitis. Interleukin-10 (IL-10) is a critical regulator of this, yet its cellular targets and mechanisms of action are not resolved. We address this here. Mice with a macrophage-selective deletion of IL-10Rα (IL-10RαMdel) developed markedly enhanced dextran sodium sulfate (DSS)-induced colitis that did not significantly differ from disease in IL-10−/− or IL-10Rα−/− mice; no impact of IL-10Rα deficiency in other lineages was observed. IL-10RαMdel colitis was associated with increased mucosal barrier disruption in the setting of intact epithelial regeneration. Lamina propria macrophages (LPMφs) did not show numerical or phenotypic differences from controls, or a competitive advantage over wild-type cells. Proinflammatory cytokine production, and particularly tumor necrosis factor-α (TNF-α), was increased, although TNF-α neutralization failed to reveal a defining role for this cytokine in the aggravated disease. Rather, IL-10RαMdel LPMφs produced substantially greater levels of nitric oxide (NO) and reactive oxygen species (ROS) than controls. Inhibition of these had modest effects in wild-type mice, although they dramatically reduced colitis severity in IL-10RαMdel mice, and largely eliminated the differential effect of DSS in them. Therefore, the palliative actions of IL-10 in DSS-induced colitis predominantly results from its macrophage-specific effects. Downregulation of NO and ROS production are central to the protective actions of IL-10.

A mouse model of clonal CD8+ T lymphocyte-mediated alopecia areata progressing to alopecia universalis

Alopecia areata is among the most prevalent autoimmune diseases, yet compared with other autoimmune conditions, it is not well studied. This in part results from limitations in the C3H/HeJ mouse and DEBR rat model systems most commonly used to study the disease, which display a low frequency and late onset. We describe a novel high-incidence model for spontaneous alopecia areata. The 1MOG244 T cell expresses dual TCRA chains, one of which, when combined with the single TCRB present, promotes the development of CD8+ T cells with specificity for hair follicles. Retroviral transgenic mice expressing this TCR develop spontaneous alopecia areata at nearly 100% incidence. Disease initially follows a reticular pattern, with regionally cyclic episodes of hair loss and regrowth, and ultimately progresses to alopecia universalis. Alopecia development is associated with CD8+ T cell activation, migration into the intrafollicular region, and hair follicle destruction. The disease may be adoptively transferred with T lymphocytes and is class I and not class II MHC-dependent. Pathologic T cells primarily express IFNG and IL-17 early in disease, with dramatic increases in cytokine production and recruitment of IL-4 and IL-10 production with disease progression. Inhibition of individual cytokines did not significantly alter disease incidence, potentially indicating redundancy in cytokine responses. These results therefore characterize a new high-incidence model for alopecia areata in C57BL/6J mice, the first to our knowledge to apply a monoclonal TCR, and indicate that class I MHC-restricted CD8+ T lymphocytes can independently mediate the pathologic response.

Retrogenic modeling of experimental allergic encephalomyelitis associates T cell frequency but not TCR functional affinity with pathogenicity.

The properties of a self-specific T cell’s TCR that determine its pathogenicity are not well understood. We developed TCR retroviral transgenic, or retrogenic, models of myelin oligodendroglial glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) to compare the pathologic potential of five H-2 Ab/MOG35–55-specific TCRs. The TCRs were cloned and retrovirally transduced into either TCRαβ-deficient hybridoma cells or Rag1−/− bone marrow progenitor cells. Comparison of the hybridomas, identical except for TCR sequence, revealed distinct responsiveness, or functionally determined affinity, for cognate Ag. Retrogenic mice were produced by transfer of transduced progenitor cells into Rag1−/− recipients. T cells were detected within 4 wk. Engraftment levels varied considerably among the different TCRs and showed separate variability among individual mice. T cells were predominantly naive and virtually exclusively CD4+ and CD25−. Relative responses of the retrogenic T cells to Ag paralleled those of the hybridoma cells. Induction of EAE through active immunization led to rapid and severe disease in all mice expressing MOG-specific TCR. The mice additionally developed spontaneous disease, the incidence of which varied with the individual receptors. Interestingly, spontaneous disease frequency and intensity could not be correlated with the functional affinity of the respective TCR. Instead, it was associated with engraftment level, even when measured weeks before the onset of disease symptoms. Our results demonstrate the feasibility of using retrogenic modeling to compare TCRs in the EAE system. They further suggest that affinity is not a primary determinant in spontaneous EAE development in mice expressing monotypic TCRs and that autoreactive T cell frequency is of greater significance.

IL-10-Dependent Suppression of Experimental Allergic Encephalomyelitis by Th2-Differentiated, Anti-TCR Redirected T Lymphocytes

We previously showed that transgenically expressed chimeric Ag-MHC-ζ receptors can Ag-specifically redirect T cells against other T cells. When the receptor’s extracellular Ag-MHC domain engages cognate TCR on an Ag-specific T cell, its cytoplasmic ζ-chain stimulates the chimeric receptor-modified T cell (RMTC). This induces effector functions such as cytolysis and cytokine release. RMTC expressing a myelin basic protein (MBP) 89–101-IAs-ζ receptor can be used therapeutically, Ag-specifically treating murine experimental allergic encephalomyelitis (EAE) mediated by MBP89-101-specific T cells. In initial studies, isolated CD8+ RMTC were therapeutically effective whereas CD4+ RMTC were not. We re-examine here the therapeutic potential of CD4+ RMTC. We demonstrate that Th2-differentiated, though not Th1-differentiated, CD4+ MBP89–101-IAs-ζ RMTC prevent actively induced or adoptively transferred EAE, and treat EAE even after antigenic diversification of the pathologic T cell response. The Th2 RMTC both Th2-deviate autoreactive T cells and suppress autoantigen-specific T cell proliferation. IL-10 is critical for the suppressive effects. Anti-IL-10R blocks RMTC-mediated modulation of EAE and suppression of autoantigen proliferation, as well as the induction of IL-10 production by autoreactive T cells. In contrast to IL-10, IL-4 is required for IL-4 production by, and hence Th2 deviation of autoreactive T cells, but not the therapeutic activity of the RMTC. These results therefore demonstrate a novel immunotherapeutic approach for the Ag-specific treatment of autoimmune disease with RMTC. They further identify an essential role for IL-10, rather than Th2-deviation itself, in the therapeutic effectiveness of these redirected Th2 T cells.

Subtle Affinity-Enhancing Mutations in a Myelin Oligodendrocyte Glycoprotein-Specific TCR Alter Specificity and Generate New Self-Reactivity

We describe a simple iterative approach to augment TCR affinity, which we studied using a myelin oligodendrocyte glycoprotein-specific TCR. We hypothesized that single amino acid modifications in TCR CDR3 could enhance TCR sensitivity through focal interactions with antigenic peptide while minimizing the risk of cross-reactivity observed previously in TCR more broadly mutagenized using in vitro evolution techniques. We show that this iterative method can indeed generate TCR with Ag sensitivity 100-fold greater than the parental receptor and can endow TCR with coreceptor independence. However, we also find that single amino acid mutations in the CDR3 can alter TCR fine specificity, affecting recognition requirements for Ag residues over most of the length of the MHC binding groove. Furthermore, minimal changes in surface-exposed CDR3 amino acids, even the addition of a single hydroxyl group or conversion of a methyl or sulfhydryl moiety to a hydroxyl, can confer modified Ag-specific TCR with new self-reactivity. In vivo modeling of modified TCR through retroviral TCR gene transfer into Rag−/− mice confirmed the biological significance of these altered reactivities, although it also demonstrated the feasibility of producing Ag-specific, positively selecting, coreceptor-independent receptors with markedly increased Ag sensitivity. These results affirm the possibility of readily generating affinity-enhanced TCR for therapeutic purposes but demonstrate that minimal changes in TCR CDR3 structure can promote self reactivity and thereby emphasize the importance of caution in validating receptors with even subtle alterations before clinical application.

Involvement of NKR-P2/NKG2D in DC-mediated killing of tumor targets: indicative of a common, innate, target-recognition paradigm?

DC are the most efficient antigen‐presenting cells that regulate the immune response. Here, we demonstrate the expression of NK cell receptor protein‐2 (NKR‐P2) on rat and mouse DC, and we show that NKR‐P2 gets reorganized upon antigen contact. DC activated with anti‐NKR‐P2 mAb exhibit enhanced apoptotic killing of tumor targets, whereas blocking the interaction between NKR‐P2 and its ligand with rNKR‐P2 abrogated apoptotic killing, suggesting NKR‐P2 to function as an activating molecule on DC. In vivo injection of anti‐NKR‐P2 mAb augmented DC activity and delayed tumor progression. NKR‐P2 signaling involved Ca2+ influx, culminating in the expression of the apoptosis‐inducing molecule, TNF‐α. Taken together, these observations suggest that NKR‐P2 (the rat orthologue of human NKG2D) acts as a target‐recognition molecule on DC.

Rational Design of T Cell Receptors with Enhanced Sensitivity for Antigen

Enhancing the affinity of therapeutic T cell receptors (TCR) without altering their specificity is a significant challenge for adoptive immunotherapy. Current efforts have primarily relied on empirical approaches. Here, we used structural analyses to identify a glycine-serine variation in the TCR that modulates antigen sensitivity. A G at position 107 within the CDR3β stalk is encoded within a single mouse and human TCR, TRBV13-2 and TRBV12-5 respectively. Most TCR bear a S107. The S hydroxymethyl side chain intercalates into the core of the CDR3β loop, stabilizing it. G107 TRBV possess a gap in their CDR3β where this S hydroxymethyl moiety would fit. We predicted based on modeling and molecular dynamics simulations that a G107S substitution would increase CDR3β stability and thereby augment receptor sensitivity. Experimentally, a G107S replacement led to an ∼10–1000 fold enhanced antigen sensitivity in 3 of 4 TRBV13-2+ TCR tested. Analysis of fine specificity indicated a preserved binding orientation. These results support the feasibility of developing high affinity antigen specific TCR for therapeutic purposes through the identification and manipulation of critical framework residues. They further indicate that amino acid variations within TRBV not directly involved in ligand contact can program TCR sensitivity, and suggest a role for CDR3 stability in this programming.

The T Cell Response to IL-10 Alters Cellular Dynamics and Paradoxically Promotes Central Nervous System Autoimmunity

IL-10 is a critical anti-inflammatory cytokine, the deficiency of which leads to spontaneous autoimmunity. However, therapeutically administered or ectopically expressed IL-10 can either suppress or promote disease. Distinct lineage-specific activities may explain the contradictory effects of IL-10. To dissect the T cell-specific response to IL-10 during organ-specific autoimmunity, we generated mice with a selective deletion of IL-10Rα in T cells and analyzed its effects in an autoimmune model, experimental allergic encephalomyelitis (EAE). Surprisingly, the T cell response to IL-10 increased EAE severity. This did not result from altered T cell functional potential; T cell cytokine profile was preserved. IL-10 also diminished the proliferation of T cells in situ within the target organ, an effect that would be expected to restrain disease. However, IL-10 acted cell autonomously to sustain the autoreactive T cells essential for immunopathogenesis, promoting their accumulation and distorting the regulatory and effector T cell balance. Indeed, in chimeric mice and after adoptive transfer, wild type T cells showed a competitive advantage over cells deficient in IL-10Rα. Therefore, T cell specific actions of IL-10 can support autoimmune inflammation, and this appears to result from an overall increase in the long term fitness of pathologic T cells. Lineage-restricted, disease-promoting activities of IL-10 should be considered in the therapeutic manipulation of the IL-10 pathway.

Correction: Differential T Cell Cytokine Receptivity and Not Signal Quality Distinguishes IL-6 and IL-10 Signaling during Th17 Differentiation.

How a large number of cytokines differentially signal through a small number of signal transduction pathways is not well resolved. This is particularly true for IL-6 and IL-10, which act primarily through STAT3 yet induce dissimilar transcriptional programs leading alternatively to pro- and anti-inflammatory effects. Kinetic differences in signaling, sustained to IL-10 and transient to IL-6, are critical to this in macrophages. T cells are also key targets of IL-6 and IL-10, yet how differential signaling in these cells leads to divergent cellular fates is unclear. We show that, unlike for macrophages, signal duration cannot explain the distinct effects of these cytokines in T cells. Rather, naive, activated, activated-rested, and memory CD4+ T cells differentially express IL-6 and IL-10 receptors in an activation state–dependent manner, and this impacts downstream cytokine effects. We show a dominant role for STAT3 in IL-6–mediated Th17 subset maturation. IL-10 cannot support Th17 differentiation because of insufficient cytokine receptivity rather than signal quality. Enforced expression of IL-10Rα on naive T cells permits an IL-10–generated STAT3 signal equivalent to that of IL-6 and equally capable of promoting Th17 formation. Similarly, naive T cell IL-10Rα expression also allows IL-10 to mimic the effects of IL-6 on both Th1/Th2 skewing and Tfh cell differentiation. Our results demonstrate a key role for the regulation of receptor expression rather than signal quality or duration in differentiating the functional outcomes of IL-6 and IL-10 signaling, and identify distinct signaling properties of these cytokines in T cells compared with myeloid cells.

PI3K orchestration of the in vivo persistence of chimeric antigen receptor-modified T cells

In vivo persistence of chimeric antigen receptor (CAR)-modified T cells correlates with therapeutic efficacy, yet CAR-specific factors that support persistence are not well resolved. Using a CD33-specific CAR in an acute myeloid leukemia (AML) model, we show how CAR expression alters T cell differentiation in a ligand independent manner. Ex vivo expanded CAR-T cells demonstrated decreased naïve and stem memory populations and increased effector subsets relative to vector-transduced control cells. This was associated with reduced in vivo persistence. Decreased persistence was not due to specificity or tumor presence, but to pre-transfer tonic signaling through the CAR CD3ζ ITAMs. We identified activation of the PI3K pathway in CD33 CAR-T cells as responsible. Treatment with a PI3K inhibitor modulated the differentiation program of CAR-T cells, preserved a less differentiated state without affecting T cell expansion, and improved in vivo persistence and reduced tumor burden. These results resolve mechanisms by which tonic signaling of CAR-T cells modulates their fate, and identifies a novel pharmacologic approach to enhance the durability of CAR-T cells for immunotherapy.