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The specific communication of multiple cell types in the tumor microenvironment plays a critical role in cancer progression. Current engineering methods have failed to adequately replicate the complexities of the tumor microenvironment (TME). In particular, generating engineered tissue-like environments with multiple TME cell types has remained challenging. Here we demonstrate the capability to pattern complex single cell circuit configurations, using a novel microfluidic bioprinting method, to study cell-cell communication in the early TME. A microfluidic dispenser (Biopixlar, Fluicell AB) was optimized to determine the delivery pressure (5 – 80 mbar), internal vacuum (0 – 80 -mbar), and external vacuum (0 – 80 -mbar) to enable highly controllable deposition of single cells suspended in complete media supplemented with polyethylene glycol (15 mg/mL, 1:1) at 1 × 106 cells/mL. Flow conditions were optimized for human cells: MDA-MB-231, MCF7, PC3, breast epithelial cells (MCF10a), fibroblasts, cancer associated fibroblasts, THP-1 derived macrophages, CD4+ T cells, CD8+ T cells, human umbilical vein endothelial cells (HUVECs), and mesenchymal stem cells. As proof of concept, the optimized settings were used to replicate a 2D tumor biopsy region of interest with high spatial precision. Next, cell-cell communication circuits were fabricated with cancer cells (PC3 or MDA-MB-231) and HUVECs. Communication circuits were bioprinted as 4 by 4 cell arrays, with 100 µm spacing between each cell, equal number of HUVECs and cancer cells, and three different cellular arrangements: alternating cell types, like cell types grouped, and groups of four like cell types. The circuits were live cell imaged for up to 30 hours to observe cell migration patterns, proliferation, and morphological changes as a function of cell-cell communication circuit arrangements. Optimal printing parameters were identified as 80 mbar delivery pressure, -25 mbar internal vacuum, and -55 mbar external vacuum. These parameters maintained >99% cell viability and ±10 µm spatial precision of printed cells. Live cell imaging of circuits containing PC3s or MDA-MB-231s with HUVECs on collagen substrates revealed changes in migration patterns, proliferation, and morphology depending on the surrounding cellular arrangement. HUVECs were highly migratory throughout the duration of the experiment, frequently extended protrusions towards nearby HUVECs, but did not display the same level of interaction with PC3s as they did with MDA-MB-231s. In MDA-MB-231 circuits, irrespective of patterning, we identified clear tendencies of HUVECs to herd MDA-MB-231s, travel overtop of MDA-MB-231s, collect and carry visible particles released from MDA-MB-231s, and maintain dendritic morphology instead of undergoing the expected vascular tubulogenesis. We found that HUVECs had the best morphology when clustered in groups of four and proliferated most when surrounded by MDA-MB-231s (alternating pattern). We found that MDA-MB-231s only proliferated when surrounded by HUVECs and had the least displacement when surrounded by like cells. These results demonstrate a method to precisely bioprint single cell circuits, enabling the investigation of cellular spatial organization and composition within the tumor microenvironment as it relates to tumor initiation and progression. Citation Format: Haylie R. Helms, Alexander E. Davies, Rebekka Duhen, Joshua M. Moreau, Ellen M. Langer, Luiz E. Bertassoni. Single cell bioprinted cell circuits for the systematic assessment of cell-cell communication in the early tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB161.

BackgroundExpression of CD103 and CD39 has been found to pinpoint tumor-reactive CD8+T cells in a variety of solid cancers. We aimed to investigate whether these markers specifically identify neoantigen-specific T cells in colorectal cancers (CRCs) with low mutation burden.Experimental designWhole-exome and RNA sequencing of 11 mismatch repair-proficient (MMR-proficient) CRCs and corresponding healthy tissues were performed to determine the presence of putative neoantigens. In parallel, tumor-infiltrating lymphocytes (TILs) were cultured from the tumor fragments and, in parallel, CD8+T cells were flow-sorted from their respective tumor digests based on single or combined expression of CD103 and CD39. Each subset was expanded and subsequently interrogated for neoantigen-directed reactivity with synthetic peptides. Neoantigen-directed reactivity was determined by flow cytometric analyses of T cell activation markers and ELISA-based detection of IFN-γ and granzyme B release. Additionally, imaging mass cytometry was applied to investigate the localization of CD103+CD39+cytotoxic T cells in tumors.ResultsNeoantigen-directed reactivity was only encountered in bulk TIL populations and CD103+CD39+(double positive, DP) CD8+T cell subsets but never in double-negative or single-positive subsets. Neoantigen-reactivity detected in bulk TIL but not in DP CD8+T cells could be attributed to CD4+T cells. CD8+T cells that were located in direct contact with cancer cells in tumor tissues were enriched for CD103 and CD39 expression.ConclusionCoexpression of CD103 and CD39 is characteristic of neoantigen-specific CD8+T cells in MMR-proficient CRCs with low mutation burden. The exploitation of these subsets in the context of adoptive T cell transfer or engineered T cell receptor therapies is a promising avenue to extend the benefits of immunotherapy to an increasing number of CRC patients.

The implantation of cancer cell lines into immunocompetent mice acts as a vaccination event, resulting in T cell immunity that impacts both tumor progression and the response to subsequent therapy. We have previously shown that blocking immunity at tumor implantation using anti-CD40L antibodies blocks development of an intratumoral antigen-specific CD8 T cell population with tissue resident memory (Trm) phenotypes, and also limits responsiveness to radiation therapy and immunotherapy combinations. Using pancreatic tumors derived from Pdx1-Cre and Pdx1-Cre /R26LSL-LSIY mice crossed with KrasLSL-G12D/+Tp53LSL-R172H/+ mice, we explored the role of CD4 T cells in providing CD40-CD40L help to generate Trm and conventional T central memory CD8 T cells (Tcm) cells in the tumor and systemically. Using depleting antibodies, we identified that Trm cells in the tumor could form independently of CD4 T cells but were lost following CD40L blockade. By contrast, systemically circulating Tcm cells that could re-expand after antigen-specific challenge were dependent on both CD4 T cells and CD40-CD40L interactions. Notably, concomitant immunity generated by tumor implantation that can prevent tumor growth on the opposite flank could also occur independently of CD4 T cells, but was lost on CD40L blockade. Systemic vaccination with Listeria monocytogenes expressing SIY efficiently induced the formation of SIY-specific Tcm cells but did not impact tumor implantation. These data suggest that tumor implantation generates Trm cells independently of CD4 T cells, and that these cells, not Tcm cells, sustain concomitant immunity. These data may impact immune therapies designed to control metastatic seeding in patients. Supported by grants from NIH (R01CA182311, R01CA244142, R01CA208644).

CD4 T helper (Th) cells play a key role in orchestrating immune responses, but the identity of the CD4 Th cells involved in the immune response against cancer remains to be defined. We analyzed the immune cell infiltrates of head and neck squamous cell carcinoma and microsatellite stable colorectal cancers and identified a subset of CD4 Th cells distinct from FOXP3+ regulatory T cells that co-express PD-1 and ICOS. This cell population, which was present in the tumor but absent in the periphery, features of chronic stimulation and displays characteristics of tissue-resident memory T cells. PD-1+ICOS+ tumor-infiltrating (TIL) CD4 Th cells are located primarily in the tumor stroma in proximity to MHC class II+ cells and are proliferating, suggesting local antigen recognition. The T-cell receptor repertoire of the PD-1+ICOS+ CD4 Th TIL is oligoclonal, with T-cell clones expanded in the tumor, but present at low frequencies in the periphery. Finally, these PD-1+ICOS+ CD4 Th TIL were shown to recognize both tumor-associated antigens and tumor-specific neoantigens, which were distinct from the epitopes recognized by the CD8 T cells from the same patients. Our findings provide an approach for isolating tumor-reactive CD4 Th TIL directly ex vivo and imply that harnessing the cooperation between CD4 and CD8 T cells might lead to more efficient tumor recognition and control in cancer patients. Supported by the Providence Portland Medical Foundation and the Developmental Research Program for Head and Neck Cancer.

Despite the success of checkpoint blockade in some cancer patients, there is an unmet need to improve outcomes. Targeting alternative pathways, such as costimulatory molecules (e.g. OX40, GITR, and 4-1BB), can enhance T cell immunity in tumor-bearing hosts. Here we describe the results from a phase Ib clinical trial (NCT02274155) in which 17 patients with locally advanced head and neck squamous cell carcinoma (HNSCC) received a murine anti-human OX40 agonist antibody (MEDI6469) prior to definitive surgical resection. The primary endpoint was to determine safety and feasibility of the anti-OX40 neoadjuvant treatment. The secondary objective was to assess the effect of anti-OX40 on lymphocyte subsets in the tumor and blood. Neoadjuvant anti-OX40 was well tolerated and did not delay surgery, thus meeting the primary endpoint. Peripheral blood phenotyping data show increases in CD4+ and CD8+ T cell proliferation two weeks after anti-OX40 administration. Comparison of tumor biopsies before and after treatment reveals an increase of activated, conventional CD4+ tumor-infiltrating lymphocytes (TIL) in most patients and higher clonality by TCRβ sequencing. Analyses of CD8+ TIL show increases in tumor-antigen reactive, proliferating CD103+ CD39+ cells in 25% of patients with evaluable tumor tissue (N = 4/16), all of whom remain disease-free. These data provide evidence that anti-OX40 prior to surgery is safe and can increase activation and proliferation of CD4+ and CD8+ T cells in blood and tumor. Our work suggests that increases in the tumor-reactive CD103+ CD39+ CD8+ TIL could serve as a potential biomarker of anti-OX40 clinical activity., Different neoadjuvant therapies have been proposed to improve immunotherapy for cancer treatment. Here, the authors perform a phase Ib clinical trial where an agonist OX40 antibody provided prior to surgery is well tolerated and increases proliferation and activation of tumor antigen-specific T cells in head and neck cancer patients.

Identifying tumor antigen-specific T cells from cancer patients has important implications for immunotherapy diagnostics and therapeutics. Here, we show that CD103+CD39+ tumor-infiltrating CD8 T cells (CD8 TIL) are enriched for tumor-reactive cells both in primary and metastatic tumors. This CD8 TIL subset is found across six different malignancies and displays an exhausted tissue-resident memory phenotype. CD103+CD39+ CD8 TILs have a distinct T-cell receptor (TCR) repertoire, with T-cell clones expanded in the tumor but present at low frequencies in the periphery. CD103+CD39+ CD8 TILs also efficiently kill autologous tumor cells in a MHC-class I-dependent manner. Finally, higher frequencies of CD103+CD39+ CD8 TILs in patients with head and neck cancer are associated with better overall survival. Our data thus describe an approach for detecting tumor-reactive CD8 TILs that will help define mechanisms of existing immunotherapy treatments, and may lead to future adoptive T-cell cancer therapies., Identifying and enumerating tumor-specific CD8 T cells are important for assessing cancer prognosis and therapy efficacy. Here the authors show that CD39 and CD103 mark a subset of tumor-infiltrating CD8 T cells that are tumor-reactive and exhibit characteristics of exhausted or tissue-resident memory T cells.

Sphingosine-1 phosphate receptor 1 (S1P1) is critical for the egress of T and B cells out of lymphoid organs. Although S1P1 agonist fingolimod is currently used for the treatment of multiple sclerosis (MS) little is known how S1P1 signaling regulates Th17 and Treg cell homeostasis. To study the impact of S1P1 signaling on Th17 and Treg cell biology, we specifically deleted S1P1 in Th17 and Treg cells using IL-17A Cre and Foxp3 Cre mice, respectively. Deletion of S1P1 in Th17 cells conferred resistance to experimental autoimmune encephalomyelitis (EAE). On the other hand, permanent deletion of S1P1 in Treg cells resulted in autoimmunity and acute deletion rendered mice more susceptible to EAE. Importantly, our study revealed that S1P1 not only regulated the egress of Treg cells out of lymphoid organs and subsequent non-lymphoid tissue distribution but also their phenotypic diversity. Most of the Treg cells found in S1P1-deficient mice as well as MS patients on fingolimod therapy had an activated phenotype and were more prone to apoptosis, thus converted to effector Treg. Our results provide novel insight into the functions of S1P1 and potential impact of long term fingolimod use on Th17 and Treg cell biology and general health in MS patients.

Innovative treatment options are required to improve cure rates in advanced colorectal cancer patients. Immune checkpoint blockade therapy (anti-PD-1) was shown to be effective in colorectal cancers with high mutation burden (e.g., mismatch repair deficient) as antitumor reactivity is largely explained by the recognition of somatically mutated antigens (neoantigens). No immunotherapeutic strategies are currently available for patients diagnosed with low mutation burden colorectal cancer. We hypothesized that if neoantigen-reactive T-cells are present in low mutation burden patients, the latter could benefit from immunotherapeutic interventions that stimulate neoantigen recognition and the onset of a robust antitumor immune response.In order to detect neoantigens, whole exome and RNA next-generation sequencing analyses were performed in cancer and healthy tissues from five colorectal cancer patients. Corresponding neoepitopes were synthesized and tested for their ability to induce immune cell activation in T-cells isolated from the tumor tissue (TIL) and from peripheral blood. Neoantigen-specific T-cell responses were identified in the majority of patients that presented with tumors carrying 25 to 36 transcribed, non-synonymous variants. Up to six different neoantigens were recognized per tumor, which resulted in a higher detection rate than anticipated based on published data. Moreover, we discovered the merits of isolating CD39+CD103+CD8+ T-cells for detection of a broad recognition of HLA class I-restricted neoantigens. This CD39+CD103+CD8+ T-cell subset comprises the majority and a broader repertoire of neoantigen-specific T-cells compared to bulk TIL populations or lymphocytes derived from peripheral blood. In conclusion, we developed a neoantigen screening pipeline to unlock the immunogenic potential of colorectal cancers with low mutation burden. We have detected a relatively high number of neoantigens that are recognized by tumor- and/or PBMC-derived T-cells in mismatch repair proficient, low mutation burden colorectal cancer patients, and show the importance of the CD39+CD103+CD8+ T-cell subset for neoantigen-based immunotherapies. These findings warrant the further exploration of the potential to employ neoantigen-targeted therapies to improve clinical outcomes of colorectal cancer patients. Citation Format: Jitske van den Bulk, Dina Ruano, Marieke E. Ijsselsteijn, Marten Visser, Ruud van der Breggen, Koen C.M.J. Peeters, Thomas Duhen, Rebekka Duhen, Andrew D Weinberg, Sjoerd S.H. van der Burg, Els M.E. Verdegaal, Noel F. de Miranda. Successful identification of neoantigen-specific T-cell responses in low mutation burden colorectal cancers for personalized cancer vaccine development [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B094.

The immune system can recognize and destroy tumor cells through T-cell mediated mechanisms. Hence, identifying tumor antigen-specific T-cells from cancer patients and expanding them in large numbers in vitro has important implications for immunotherapy diagnostics and therapeutics. Here we show that tumor-reactive T-cells are enriched in a subset of tumor-infiltrating CD8 T-cells (CD8 TILs) identified by co-expression of CD103 and CD39 both in primary and metastatic tumors. The CD103+CD39+ CD8 TILs are present at high frequencies in melanoma and mismatch repair-deficient colon cancer but at low frequencies in microsatellite stable (MSS) colon cancer and colorectal liver metastasis. This cell population displays a distinct T-cell receptor (TCR) repertoire, with T-cell clones expanded in the tumor but present at low frequencies in the periphery. Importantly, we show in a MSS colon cancer patient that a very low number of CD103+CD39+ CD8 TILs can be expanded in vitro and that those cells recognize tumor-specific neoantigens. Finally, patients with head and neck cancer whose CD8 TILs contained a higher frequency of CD103+CD39+ cells experienced a greater overall survival. Our work suggests that CD103+CD39+ CD8 TILs are key players in the patient’s antitumor response and describe an approach for detecting and expanding those cells, which will help improve adoptive TIL therapy for cancer patients. Citation Format: Thomas Duhen, Rebekka Duhen, Ryan Montler, Tarsem Moudgil, Jitske van den Bulk, Bernard A. Fox, Shu-Ching Chang, Gary Grunkemeier, Els M.E. Verdegaal, Noel F. de Miranda, Rom Leidner, Richard B. Bell, Andrew D. Weinberg. A new strategy to identify and expand tumor-reactive CD8 TILs in human solid tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A186.

Sphingosine-1 phosphate receptor 1 (S1P1) is critical for the egress of T and B cells out of lymphoid organs. Although S1P1 agonist fingolimod is currently used for the treatment of multiple sclerosis (MS) little is known how S1P1 signaling regulates Th17 and Treg cell homeostasis. To study the impact of S1P1 signaling on Th17 and Treg cell biology, we specifically deleted S1P1 in Th17 and Treg cells using IL-17A Cre and Foxp3 Cre mice, respectively. Deletion of S1P1 in Th17 cells conferred resistance to experimental autoimmune encephalomyelitis (EAE). On the other hand, permanent deletion of S1P1 in Treg cells resulted in autoimmunity and acute deletion rendered mice more susceptible to EAE. Importantly, our study revealed that S1P1 not only regulated the egress of Treg cells out of lymphoid organs and subsequent non-lymphoid tissue distribution but also their phenotypic diversity. Most of the Treg cells found in S1P1-deficient mice as well as MS patients on fingolimod therapy had an activated phenotype and were more prone to apoptosis, thus converted to effector Treg. Our results provide novel insight into the functions of S1P1 and potential impact of long term fingolimod use on Th17 and Treg cell biology and general health in MS patients.

The neutralization of α4 integrin is currently used as treatment in several autoimmune diseases and is thought to prevent the entry of most immune cells in target tissues. In this study, we showed that selective deletion of α4 integrin in T cells did not prevent but delayed the development of experimental autoimmune encephalomyelitis. Whereas both Th1 and Th17 cells infiltrate the CNS of wild-type mice, T cells present in the CNS of mice lacking α4 integrin were mainly enriched in Th17 cells, suggesting that this T cell subset uses other integrins to access the CNS. In contrast, α4 integrin expression is important for Th1 cells to enter the CNS and for the stability of their Th1-associated genetic program. Therefore, our data suggest that anti-α4 integrin Ab treatment may be more efficient in the treatment of Th1- rather than Th17-mediated disease.

6011Background: This phase Ib clinical trial was performed to investigate an agonistic murine antibody to OX40 (MEDI6469) at various dose intervals prior to definitive surgical resection in patient...

Background: Head and neck squamous cell carcinomas (HNSCC) produce suppressive factors that impair the immune system, thus limiting effective antitumor immunity. OX40 is a member of the tumor necrosis factor (TNF) receptor family and its biologic activity leads to potent co-stimulation, which can enhance T-cell memory, proliferation and antitumor activity in patients with metastatic cancer. However, its effect on wound healing and the optimal timing of administration in relation to surgery to induce immune changes within the tumor microenvironment (TME) is not known. Objectives: To determine the safety and peak immunologic activity of neoadjuvant anti-OX40 treatment administered prior to definitive surgical resection in patients with locoregionally advanced HNSCC. Methods: Between January 2016 and July 2016, 10 patients with locoregionally advanced HNSCC were enrolled into this phase Ib neoadjuvant time course trial testing a murine antibody to OX40 (MEDI6469) administered 2 days, 1 week and 2 weeks prior to definitive surgical resection. In order to assess changes in the tumor microenvironment (TME), a tissue biopsy and peripheral blood samples were obtained prior to MEDI6469 infusion and tissue was also harvested at the time of surgical resection from the primary tumor site, metastatic and draining lymph nodes along with peripheral blood. Assessments of tumor infiltrating lymphocyte (TIL) populations were performed based on flow cytometry and fluorescent multiplex immunohistochemistry (mIHC); other circulating immunologic parameters that correlate with changes induced by MEDI6469 administration were also measured. These immune changes were assessed and compiled in a “cumulative suppression index,” which incorporates immunosuppressive elements within the tumor, such as FoxP3+ and PD-L1+ cells, to be correlated with clinical variables and outcome. Surgical complications were described using the Clavien-Dindo grading scale. Clinical trial information: NCT02274155. Results: MEDI6469 administration was well tolerated and there were no grade 3 or 4 adverse events (AEs) attributable to anti-OX40 treatment. The toxicity profile was mild, most commonly consisting of low-grade fever prior to surgery, which was performed in all patients without delay. Postoperative grade 3 and 4 complications per Clavien-Dindo scale were observed in two patients. Immunologic changes were observed at all time courses with significant activation and proliferation of CD4+ and CD8+ central and effector memory T-cell populations in both the TME and circulation occurring between 12 and 19 days following MEDI6469 infusion. Ki67 was specifically induced in the TME and on peripheral blood PBMCs after MEDI6469 administration, returning to baseline at Day 55. Up-regulation of PD-L1 was also seen in the tumor post treatment in the majority of specimens. In the tumor, expression of CD39, ICOS and PD-1 is increased on CD4+ T cells in almost all patients and a recently identified tumor-reactive T-cell subset of CD39+CD103+CD8+ T cells, with resident memory phenotype, was increased in some patients. Conclusion: Preoperative MEDI6469 administration prior to surgery is feasible and safe in patients with HNSCC and results in activation and proliferation of T cell populations and up-regulation of PD-L1 in tumor cells occurring between 12 and 19 days following infusion. Citation Format: R. Bryan Bell, Rom S. Leidner, Rebekka A. Duhen, Carmen Ballesteros-Merino, Zipei Feng, Yoshinobu Koguchi, Carlo B. Bifulco, Brendan D. Curti, Walter J. Urba, Bernard A. Fox, Andrew D. Weinberg. Anti-OX40 (MEDI6469) prior to definitive surgical resection in patients with head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 37.

Experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS), results from an autoimmune attack of the central nervous system (CNS) by effector T helper (Th) 1 and Th17 cells. Regulatory T cells (Treg) can control effector T cells and limit the progression of CNS autoimmunity. Integrin alpha 4 (Itga4) is critical for the entry of Th1 but not Th17 cells into the CNS during EAE. Whether Itga4 controls the homing of Tregs in the CNS and whether Tregs can limit Th17-mediated EAE has, however, not been addressed. Through selective elimination of Itga4 in Foxp3-expressing cells, we show here that Tregs can suppress Th17-mediated EAE and enter into the CNS independently of Itga4. Furthermore, similarly to Th17 cells and in contrast to Th1 cells, Tregs depend on LFA-1 for their entry into the CNS in the absence of Itga4. Therefore, these data suggest that the efficacy of Itga4 neutralization on MS progression may be associated with the prevention of Th1 cells and the maintenance of Tregs migration into the CNS.

IL-17–producing CD4+ T (Th17) cells, along with IFN-γ–expressing Th1 cells, represent two major pathogenic T cell subsets in experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). The cytokines and transcription factors involved in the development and effector functions of Th1 and Th17 cells have been largely characterized. Among them, IL-23 is essential for the generation of stable and encephalitogenic Th17 cells and for the development of EAE. The IL-7/IL-7R signaling axis participates in cell survival, and perturbation of this pathway has been associated with enhanced susceptibility to MS. A link between IL-23–driven pathogenic T cells and IL-7/IL-7R signaling has previously been proposed, but has not been formally addressed. In the current study, we showed that Th17 cells from mice with EAE express high levels of IL-7Rα compared with Th1 cells. Using mice that constitutively express IL-7Rα on T cells, we determined that sustained IL-7R expression in IL-23R–deficient mice could not drive pathogenic T cells and the development of EAE. IL-7 inhibited the differentiation of Th17 cells, but promoted IFN-γ and GM-CSF secretion in vitro. In vivo IL-7/anti–IL-7 mAb complexes selectively expanded and enhanced the proliferation of CXCR3-expressing Th1 cells, but did not impact Th17 cells and EAE development in wild-type and IL-23R–deficient mice. Importantly, high IL-7 expression was detected in the CNS during EAE and could drive the plasticity of Th17 cells to IFN-γ–producing T cells. Together, these data address the contribution of IL-23/IL-23R and IL-7/IL-7R signaling in Th17 and Th1 cell dynamics during CNS autoimmunity.

During the development of experimental autoimmune encephalomyelitis (EAE), the proportion of pathogenic and myelin-specific cells within CNS-infiltrating cytokine-producing Th cells is unknown. Using an IL-17A/IFN-γ double reporter mouse and I-Ab/myelin oligodendrocyte glycoprotein 38–49 tetramer, we show in this study that IL-17+IFN-γ+ Th cells, which are expanded in the CNS during EAE, are highly enriched in myelin oligodendrocyte glycoprotein–specific T cells. We further demonstrate that IL-23 is essential for the generation and expansion of IFN-γ–producing Th17 cells independently of the Th1-associated transcription factors T-bet, STAT1, and STAT4. Furthermore, Th17 and IL-17+IFN-γ+ Th cells can induce CNS autoimmunity independently of T-bet. Whereas T-bet is crucial for Th1-mediated EAE, it is dispensable for Th17 cell–mediated autoimmunity. Our results suggest the existence of different epigenetic programs that regulate IFN-γ expression in Th1 and Th17 cells.

FOXP3+ regulatory T (Treg) cells are a broadly acting and potent anti-inflammatory population of CD4+ T cells essential for maintaining immune homeostasis and preventing debilitating autoimmunity. Based on chemokine receptor expression, we identified distinct populations of Treg cells in human blood expected to colocalize with different Th cell subsets. Although each population was functionally suppressive, they displayed unique patterns of pro- and anti-inflammatory cytokine production, differentially expressed lineage-specifying transcription factors, and responded differently to antigens associated with Th1 and Th17 responses. These results highlight a previously unappreciated degree of phenotypic and functional diversity in human Treg cells that allows subsets with unique specificities and immunomodulatory functions to be targeted to defined immune environments during different types of inflammatory responses.

The integrin alpha 4 (Itga4) is expressed on immune cells and is an important molecule for the migration of T cells in various organs. Because of this propriety, a monoclonal antibody specific to Itga4 is used for the treatment of multiple sclerosis (MS). Although effective, anti-Itga4 therapy has been associated with the development of progressive multifocal leukoencephalopathy (PML), a life threatening condition, which results from a rare infection of the central nervous system (CNS) by the JC virus. Despite its use and efficacy in the clinic, it remains unclear how different cells of the adaptive immune system are affected by Itga4 deletion or neutralization. Experimental autoimmune encephalomyelitis (EAE) serves as an animal model of multiple sclerosis (MS). It results from an autoimmune attack by myelin reactive effector T helper (Th) 1 and Th17 cells and progressive demyelination. Foxp3+ regulatory T cells can control effector T cell responses but are not always effective during autoimmune disease progression. Using mice with selective deletion of Itga4 in T cells, we previously reported that Itga4 selectively inhibits the migration of Th 1 cells but not Th17 cells in the CNS. We have now established that Itga4 differentially affect the migration of effector and regulatory T cells during EAE. The implication of our findings for MS therapy will be discussed.

It is now established that T helper (Th) cell subsets, particularly Th17 cells, are more dynamic than originally anticipated and can acquire the expression of other lineage effector cytokines. However, the molecular cues that dictate their plasticity are unknown. We show that IL-23 plays a central role in the plasticity of Th17 cells and is essential for the generation and propagation of IFN-γ-producing Th17 (IL-17+ IFN-γ+) cells. During the development of experimental autoimmune encephalomyelitis (EAE) IL-17+ IFN-γ+ T cells are preferentially expanded in the central nervous system (CNS). Using an IL-17A-IFN-γ double reporter mouse and I-Ab/MOG38-49 tetramer, we determined that they displayed characteristics of pathogenic T cells, such as GM-CSF expression, and contained the highest number of MOG-specific T cells. Interestingly, the Th1-associated transcription factors T-bet, STAT1 and STAT4 were not involved in the induction of IFN-γ by Th17 cells in vitro. Moreover, in contrast to previous studies showing a role for T-bet in Th1 and Th17-mediated EAE, we demonstrate that T-bet expression in T cells is not required for the emergence of CNS-infiltrating IL-17+ IFN-γ+ T cells and the development of Th17-driven EAE. Our results suggest the existence of different epigenetic programs that regulate IFN-γ expression in Th1 and Th17 cells.