Emerging interleukin targets in the tumour microenvironment: implications for the treatment of gastrointestinal tumours.

The effectiveness of antitumour immunity is dependent on intricate cytokine networks. Interleukins (ILs) are important mediators of complex interactions within the tumour microenvironment, including regulation of tumour-infiltrating lymphocyte proliferation, differentiation, migration and activation. Our evolving and increasingly nuanced understanding of the cell type-specific and heterogeneous effects of IL signalling has presented unique opportunities to fine-tune elaborate IL networks and engineer new targeted immunotherapeutics. In this review, we provide a primer for clinicians on the challenges and potential of IL-based treatment. We specifically detail the roles of IL-2, IL-10, IL-12 and IL-15 in shaping the tumour-immune landscape of gastrointestinal malignancies, paying particular attention to promising preclinical findings, early-stage clinical research and innovative therapeutic approaches that may properly place ILs to the forefront of immunotherapy regimens.

Blockade of interleukin 10 potentiates antitumour immune function in human colorectal cancer liver metastases.

OBJECTIVE: Programmed cell death protein 1 (PD-1) checkpoint inhibition and adoptive cellular therapy have had limited success in patients with microsatellite stable colorectal cancer liver metastases (CRLM). We sought to evaluate the effect of interleukin 10 (IL-10) blockade on endogenous T cell and chimeric antigen receptor T (CAR-T) cell antitumour function in CRLM slice cultures. DESIGN: We created organotypic slice cultures from human CRLM (n=38 patients' tumours) and tested the antitumour effects of a neutralising antibody against IL-10 (αIL-10) both alone as treatment and in combination with exogenously administered carcinoembryonic antigen (CEA)-specific CAR-T cells. We evaluated slice cultures with single and multiplex immunohistochemistry, in situ hybridisation, single-cell RNA sequencing, reverse-phase protein arrays and time-lapse fluorescent microscopy. RESULTS: αIL-10 generated a 1.8-fold increase in T cell-mediated carcinoma cell death in human CRLM slice cultures. αIL-10 significantly increased proportions of CD8+ T cells without exhaustion transcription changes, and increased human leukocyte antigen - DR isotype (HLA-DR) expression of macrophages. The antitumour effects of αIL-10 were reversed by major histocompatibility complex class I or II (MHC-I or MHC-II) blockade, confirming the essential role of antigen presenting cells. Interrupting IL-10 signalling also rescued murine CAR-T cell proliferation and cytotoxicity from myeloid cell-mediated immunosuppression. In human CRLM slices, αIL-10 increased CEA-specific CAR-T cell activation and CAR-T cell-mediated cytotoxicity, with nearly 70% carcinoma cell apoptosis across multiple human tumours. Pretreatment with an IL-10 receptor blocking antibody also potentiated CAR-T function. CONCLUSION: Neutralising the effects of IL-10 in human CRLM has therapeutic potential as a stand-alone treatment and to augment the function of adoptively transferred CAR-T cells.

B7-H3 Specific CAR T Cells for the Naturally Occurring, Spontaneous Canine Sarcoma Model.

One obstacle for human solid tumor immunotherapy research is the lack of clinically relevant animal models. In this study, we sought to establish a chimeric antigen receptor (CAR) T-cell treatment model for naturally occurring canine sarcomas as a model for human CAR T-cell therapy. Canine CARs specific for B7-H3 were constructed using a single-chain variable fragment derived from the human B7-H3-specific antibody MGA271, which we confirmed to be cross-reactive with canine B7-H3. After refining activation, transduction, and expansion methods, we confirmed target killing in a tumor spheroid three-dimensional assay. We designed a B7-H3 canine CAR T-cell and achieved consistently high levels of transduction efficacy, expansion, and in vitro tumor killing. Safety of the CAR T cells were confirmed in two purposely bred healthy canine subjects following lymphodepletion by cyclophosphamide and fludarabine. Immune response, clinical parameters, and manifestation were closely monitored after treatments and were shown to resemble that of humans. No severe adverse events were observed. In summary, we demonstrated that similar to human cancers, B7-H3 can serve as a target for canine solid tumors. We successfully generated highly functional canine B7-H3-specific CAR T-cell products using a production protocol that closely models human CAR T-cell production procedure. The treatment regimen that we designed was confirmed to be safe in vivo. Our research provides a promising direction to establish in vitro and in vivo models for immunotherapy for canine and human solid tumor treatment.

Key chemokines direct migration of immune cells in solid tumors.

Immune cell infiltration into solid tumors, their movement within the tumor microenvironment (TME), and interaction with other immune cells are controlled by their directed migration towards gradients of chemokines. Dysregulated chemokine signaling in TME favors the growth of tumors, exclusion of effector immune cells, and abundance of immunosuppressive cells. Key chemokines directing the migration of immune cells into tumor tissue have been identified. In this review, we discuss well-studied chemokine receptors that regulate migration of effector and immunosuppressive immune cells in the context of cancer immunology. We discuss preclinical models that have described the role of respective chemokine receptors in immune cell migration into TME and review preclinical and clinical studies that target chemokine signaling as standalone or combination therapies.

IL-15 mediated expansion of rare durable memory T cells following adoptive cellular therapy.

BACKGROUND: Synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) are ideal solid tumors for the development of adoptive cellular therapy (ACT) targeting NY-ESO-1, as a high frequency of tumors homogeneously express this cancer-testes antigen. Data from early phase clinical trials have shown antitumor activity after the adoptive transfer of NY-ESO-1-specific T cells. In these studies, persistence of NY-ESO-1 specific T cells is highly correlated with response to ACT, but patients often continue to have detectable transferred cells in their peripheral blood following progression. METHOD: We performed a phase I clinical trial evaluating the safety of NY-ESO-1-specific endogenous T cells (ETC) following cyclophosphamide conditioning. Peripheral blood mononuclear cells (PBMCs) from treated patients were evaluated by flow cytometry and gene expression analysis as well as through ex vivo culture assays with and without IL-15. RESULTS: Four patients were treated in a cohort using ETC targeting NY-ESO-1 following cyclophosphamide conditioning. Treatment was well tolerated without significant toxicity, but all patients ultimately had disease progression. In two of four patients, we obtained post-treatment tumor tissue and in both, NY-ESO-1 antigen was retained despite clear detectable persisting NY-ESO-1-specific T cells in the peripheral blood. Despite a memory phenotype, these persisting cells lacked markers of proliferation or activation. However, in ex vivo culture assays, they could be induced to proliferate and kill tumor using IL-15. These results were also seen in PBMCs from two patients who received gene-engineered T-cell receptor-based products at other centers. CONCLUSIONS: ETC targeting NY-ESO-1 with single-agent cyclophosphamide alone conditioning was well tolerated in patients with SS and those with MRCL. IL-15 can induce proliferation and activity in persisting NY-ESO-1-specific T cells even in patients with disease progression following ACT. These results support future work evaluating whether IL-15 could be incorporated into ACT trials post-infusion or at the time of progression.

Innate cell microenvironments in lymph nodes shape the generation of T cell responses during type I inflammation.

Microanatomical organization of innate immune cells within lymph nodes (LNs) is critical for the generation of adaptive responses. In particular, steady-state LN-resident dendritic cells (Res cDCs) are strategically localized to intercept lymph-draining antigens. Whether myeloid cell organization changes during inflammation and how that might affect the generation of immune responses are unknown. Here, we report that during type I, but not type II, inflammation after adjuvant immunization or viral infection, antigen-presenting Res cDCs undergo CCR7-dependent intranodal repositioning from the LN periphery into the T cell zone (TZ) to elicit T cell priming. Concurrently, inflammatory monocytes infiltrate the LNs via local blood vessels, enter the TZ, and cooperate with Res cDCs by providing polarizing cytokines to optimize T cell effector differentiation. Monocyte infiltration is nonuniform across LNs, generating distinct microenvironments with varied local innate cell composition. These spatial microdomains are associated with divergent early T cell effector programming, indicating that innate microenvironments within LNs play a critical role in regulating the quality and heterogeneity of T cell responses. Together, our findings reveal that dynamic modulation of innate cell microenvironments during type I inflammation leads to optimized generation of adaptive immune responses to vaccines and infections.

Dendritic Cells in the Tumor Microenvironment.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) of the immune system. They capture foreign antigens and can present them to lymphocytes, that is, T cells and B cells, to activate them. DCs are the most potent of all immune cells at inducing the adaptive immune system. Thus, the presence of DCs at the anatomical site of the immune challenge is imperative for the immune system to mount an effective immune response. From the anatomical site of the immune challenge, DCs cargo antigens to the draining lymph nodes, specialized immune organs where adaptive immunity is generated. DCs are heterogeneous as a type of immune cell, and various subsets of DCs have been reported and their functions described. In this chapter, we discuss various aspects of DC development and function. We further discuss how various tumor microenvironments can affect DC development, function, and migration, thus evading a strong adaptive immune response.

Histiocyte predominant myocarditis resulting from the addition of interferon gamma to cyclophosphamide-based lymphodepletion for adoptive cellular therapy.

BACKGROUND: Adoptive cellular therapy (ACT) is a promising treatment for synovial sarcoma (SS) with reported response rates of over 50%. However, more work is needed to obtain deeper and more durable responses. SS has a 'cold' tumor immune microenvironment with low levels of major histocompatibility complex (MHC) expression and few T-cell infiltrates, which could represent a barrier toward successful treatment with ACT. We previously demonstrated that both MHC expression and T-cell infiltration can be increased using systemic interferon gamma (IFN-γ), which could improve the efficacy of ACT for SS. CASE PRESENTATION: We launched a phase I trial incorporating four weekly doses of IFN-γ in an ACT regimen of high-dose cyclophosphamide (HD Cy), NY-ESO-1-specific T cells, and postinfusion low-dose interleukin (IL)-2. Two patients were treated. While one patient had significant tumor regression and resultant clinical benefit, the other patient suffered a fatal histiocytic myocarditis. Therefore, this cohort was terminated for safety concerns. CONCLUSION: We describe a new and serious toxicity of immunotherapy from IFN-γ combined with HD Cy-based lymphodepletion and low-dose IL-2. While IFN-γ should not be used concurrently with HD Cy or with low dose IL-2, IFN-γ may still be important in sensitizing SS for ACT. Future studies should avoid using IFN-γ during the immediate period before/after cell infusion. TRIAL REGISTRATION NUMBERS: NCT04177021, NCT01957709, and NCT03063632.

Efficient homing of T cells via afferent lymphatics requires mechanical arrest and integrin-supported chemokine guidance.

Little is known regarding lymph node (LN)-homing of immune cells via afferent lymphatics. Here, we show, using a photo-convertible Dendra-2 reporter, that recently activated CD4 T cells enter downstream LNs via afferent lymphatics at high frequencies. Intra-lymphatic immune cell transfer and live imaging data further show that activated T cells come to an instantaneous arrest mediated passively by the mechanical 3D-sieve barrier of the LN subcapsular sinus (SCS). Arrested T cells subsequently migrate randomly on the sinus floor independent of both chemokines and integrins. However, chemokine receptors are imperative for guiding cells out of the SCS, and for their subsequent directional translocation towards the T cell zone. By contrast, integrins are dispensable for LN homing, yet still contribute by increasing the dwell time within the SCS and by potentially enhancing T cell sensing of chemokine gradients. Together, these findings provide fundamental insights into mechanisms that control homing of lymph-derived immune cells.

Patents and Innovation Among Neurosurgeons from the American Association of Neurological Surgeons.

Objective Neurosurgeons have taken on the role of innovators, continuing to move the field forward over the centuries. More recently, innovation has taken the form of new technological devices and therapeutics, which require patenting. The aim of this study is to identify major areas of innovation in the field of neurosurgery by evaluating patent records. Methods This study quantifies the number of patents the American Association of Neurological Surgeons (AANS) neurosurgeons hold across different subspecialties. The United States Patent and Trademark Office (USPTO) patent database was queried using the names of 7,293 AANS members who filed patents between 1976 and 2019. Results A total of 346 (4.7%) AANS neurosurgeons hold a total of 1,025 patents. The number of patents held by each neurosurgeon ranged from one to 109. The areas that patents were filed under include cellular and genetic science (40), drug delivery (45), image guidance (82), neuromodulation (52), pain (7), peripheral nerve stimulation (24), spine (398), surgical devices (148), trauma (16), tumor (78), vascular (67), and other (68). No patents were filed under pediatrics (0). The fields with the greatest number of filed patents are spine, instruments/devices, and image guidance. Conclusion Given the technical nature of the field of neurosurgery, instruments and devices that improve localization, visualization, targeting, and spinal reconstruction are often in demand. Furthermore, since the rates of spinal procedures and implants continue to increase, higher patenting may be motivated by the opportunity to develop new products that can result in royalty payments to neurosurgeons. The advent of new technologies undoubtedly continues to push the field of neurosurgery forward.

Durable tumor regression in highly refractory metastatic KIT/PDGFRA wild-type GIST following treatment with nivolumab.

Gastrointestinal stromal tumor (GIST) is a devastating disease, especially in the setting of metastasis. The natural progression of GIST has been significantly altered by the development of small molecule tyrosine kinase inhibitors (TKIs), including imatinib, sunitinib, and regorafenib, all of which are FDA approved. However, TKIs are not always well-tolerated, and the refractory disease continues to be a problem. For these reasons, alternative treatments are needed. In this report, we discuss a patient with metastatic wild-type (WT) GIST refractory to multiple TKIs, but with a durable clinical response to the anti-programmed cell death protein 1 (PD-1) antibody, nivolumab. This report suggests that continued research evaluating checkpoint inhibitors in GIST is warranted.

Snapshot: Socioeconomic Competence in US Neurosurgery Residents.

INTRODUCTION: Socioeconomic topics such as federal mandates/regulations, conflict of interest, and practice management have become increasingly important for all neurosurgeons. Graduating residents immediately need a host of skills to successfully navigate neurosurgical practice. Surgical and medical skills are closely evaluated through the American Board of Neurological Surgery, and a formal socioeconomic curriculum has been developed with defined milestones. Nevertheless, little has been done to evaluate neurosurgery resident competence in socioeconomic and medicolegal principles. The purpose of this study was to assess the competence of Accreditation Council for Graduate Medical Education neurosurgical residents in socioeconomic knowledge. METHODS: Neurosurgery resident members of the American Association of Neurological Surgeons (N = 1385) were sent a Survey Monkey of 10 questions. The survey covered the most basic of socioeconomic principles. Initial survey responses were collected across a 1-month period from April to May 2018. RESULTS: The response rate was 14% (194/1385). Overall, neurosurgery residents would have received a grade of D, with an average score of 67% on the survey. For 7 of the 10 questions, the majority (>50%) of neurosurgery residents answered correctly. Furthermore, for 3 questions, more than 90% of residents selected the correct answer. However, for one-half of all questions, residents averaged a score of less than 65%. Residents tended to answer questions correctly for physician compensation and compensation models, but incorrectly for topics of informed consent, Controlled Substances Act, and conflicts of interest. CONCLUSION: With the increasing complexity of neurosurgery practice, solid knowledge of socioeconomic topics is essential. The study confirms suspected deficiencies in socioeconomic proficiency among neurosurgery residents, despite the availability of a validated curriculum. This knowledge gap will likely affect career success and satisfaction. Nevertheless, this survey had a significantly low response rate, and it may be an incomplete representation of the neurosurgical resident mind. Focused educational initiatives through the neurosurgical Residency Review Committee and individual training programs must facilitate an action plan that ensures the effective implementation of socioeconomic curricula.

Systemic Interferon-γ Increases MHC Class I Expression and T-cell Infiltration in Cold Tumors: Results of a Phase 0 Clinical Trial.

Interferon-γ (IFNγ) has been studied as a cancer treatment with limited evidence of clinical benefit. However, it could play a role in cancer immunotherapy combination treatments. Despite high expression of immunogenic cancer-testis antigens, synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) have a cold tumor microenvironment (TME), with few infiltrating T cells and low expression of major histocompatibility complex class I (MHC-I). We hypothesized that IFNγ treatment could drive inflammation in a cold TME, facilitating further immunotherapy. We conducted a phase 0 clinical trial treating 8 SS or MRCL patients with weekly systemic IFNγ. We performed pre- and posttreatment biopsies. IFNγ changed the SS and MRCL TME, inducing tumor-surface MHC-I expression and significant T-cell infiltration (P < 0.05). Gene-expression analysis suggested increased tumor antigen presentation and less exhausted phenotypes of the tumor-infiltrating T cells. Newly emergent antigen-specific humoral and/or T-cell responses were found in 3 of 7 evaluable patients. However, increased expression of PD-L1 was observed on tumor-infiltrating myeloid cells and in some cases tumor cells. These findings suggest that systemic IFNγ used to convert SS and MRCL into "hot" tumors will work in concert with anti-PD-1 therapy to provide patient benefit.

Preoperative and Histological Predictors of Recurrence and Survival in Atypical Meningioma After Initial Gross Total Resection.

OBJECTIVE: Atypical (World Health Organization grade II) meningiomas (AMs) have been associated with a substantial risk of recurrence even after complete, gross total resection (GTR). The present study evaluated the clinical and AM tumor histopathological features that might predict for the risk of recurrence and survival within this patient population. METHODS: The data from 72 consecutive patients who had undergone primary GTR for AM from 2007 to 2016 and corresponding tumor specimens at a single institution were reviewed. The preoperative patient and tumor characteristics were correlated with the postresection outcomes, including recurrence and 1-year survival. Cox regression models on recurrence-free survival (RFS) and Kaplan-Meier survival estimates were performed. RESULTS: The overall 1-, 3-, and 5-year RFS estimates for the AM cohort were 100.0%, 82.4%, and 78.1% after resection, respectively. A high mitotic index was an independent predictor of RFS on Cox regression analysis (hazard ratio, 1.26; P = 0.008), and the tumor volume showed a trend toward a significant association (hazard ratio, 0.93; P = 0.079). Patient age and the mitotic index were significantly associated with 1-year mortality (odds ratio, 1.11 and 1.36, respectively; P = 0.028 and P = 0.045, respectively). CONCLUSIONS: AM tumors with a high proliferative index showed an increased likelihood of recurrence and short-term survival even after complete GTR. A smaller tumor volume might also have contributed to an increased risk of recurrence for patients with AM. Although other histopathological features were not linked to recurrence or mortality for patients with AM, the biopsy findings can indicate key predictive information, and further molecular analysis might reveal additional prognostic markers.

The Predictive Role of Intraoperative Visual Evoked Potentials in Visual Improvement After Endoscopic Pituitary Tumor Resection in Large and Complex Tumors: Description and Validation of a Method.

BACKGROUND: With the advent of extensive endoscopic approaches for pituitary tumors, there has also been an increase in surgery for larger and more complex tumors. Intraoperative manipulation during endoscopic resection of sellar tumors poses potential risk in postoperative visual function in this tumor population. This study proposes a method of accurate intraoperative monitoring of visual evoked potentials (VEPs) and its role in predicting visual function outcomes. METHODS: Intraoperative VEPs were monitored for 42 resections from a single surgical team, with average tumor size of 2.84 cm. Changes in VEP amplitude and latency in excess of 50% were considered significant. Preoperative and postoperative visual information was obtained from ophthalmology and hospital records, along with patient demographics, comorbidities, and tumor characteristics. RESULTS: Patients were stratified as experiencing deteriorations in VEPs that did not restore to baseline (n = 4), deteriorations in VEPs that did restore to baseline (n = 6), no change in VEPs (n = 31), and improvement in VEPs (n = 1). Correlation between VEP changes and postoperative visual fields was measured through univariate ordered logistic regression. Improved intraoperative VEP measurements were associated with odds ratio (OR) of visual field improvement of 3.15 (95% confidence interval, 1.15-8.59). Specifically, changes in VEP amplitude were positively associated with visual field improvement with OR of 4.35 (OR, 1.29-14.7). No association was observed between VEPs and other patient or tumor characteristics. CONCLUSION: Changes in VEP amplitude during endoscopic sellar tumor resection correlate with postoperative visual function. Intraoperative VEP monitoring can serve an important role in preventing postoperative visual field loss.

Malpractice Litigation in Brain Tumor Surgery: A 31-Year Analysis of Causative Factors in the United States from the Westlaw Database.

BACKGROUND: Medical malpractice litigation is an issue of major concern in neurosurgery, with 19.1% of neurosurgeons facing a claim annually. Neurosurgery possesses the greatest risk of malpractice of any specialty, likely owing to the complex clinical environment and disease severity. In the present study, we have characterized such litigation to determine the common factors that compel plaintiffs to file these claims. METHODS: WestLawNext, a prominent legal database, was used to identify all cases from 1985 to 2016 related to brain tumors. A total of 225 cases were identified, and each was analyzed for the cause of litigation (multiple causes were permitted). Because many had >1 ground for litigation, the reported percentages were based on the total counts of litigation rather than the number of cases. Additional information was collected from each case, including location, tumor type, and physician specialty. RESULTS: The cases were distributed across 36 states and U.S. territories: California (n = 42; 20%) and New York (n = 28; 13%) had the greatest number of cases. The top reasons for litigation were failure to diagnose (n = 109; 28%), failure to treat (n = 72; 18%), procedural error (n = 63; 16%), and failure to refer for diagnostic tests (n = 55; 14%). The most common diagnoses included pituitary adenoma (n = 26; 12%), acoustic neuroma (n = 27; 12%), and meningioma (n = 23; 10%). CONCLUSIONS: Malpractice litigation contributes to high overhead and physician burnout and escalates the cost of patient care. We found that benign brain tumors were the most common in litigation and that surgical issues accounted for only a small percentage.

Antidepressant use in patients with meningioma: is there an association with tumor recurrence?

OBJECTIVE With increasing general use of antidepressants (ADs), multiple studies have noted a small protective effect of ADs for patients with glioma, but their impact on meningioma has not been established. This study aims to evaluate the role of ADs in the context of additional clinical factors in relation to long-term risk of meningioma recurrence. METHODS One hundred five patients with an intracranial meningioma presenting from 2011-2014 with at least 3 years of follow-up (median 4.2 years) after resection were reviewed. AD use along with demographics, tumor characteristics, and outcomes were recorded. Multivariate logistic regression was used to analyze the association of AD use with tumor recurrence, including other clinical measures significantly associated with recurrence as covariates. RESULTS Twenty-nine patients (27.4%) were taking ADs (27 selective serotonin reuptake inhibitors, 2 norepinephrine-dopamine reuptake inhibitors) prior to tumor recurrence. Their tumors largely affected the frontal (31.0%) or parietal lobe (17.2%) and were located in convexity, parasagittal, or falcine (CPF) areas more frequently than skull base areas relative to the tumors of non-AD users (p = 0.035). AD use was found to be an independent predictor of recurrence, in addition to subtotal resection and WHO grade II/III classification (p values < 0.05). The median time from AD prescription to tumor recurrence was 36.6 months (interquartile range [IQR] = 20.9-62.9 months) and median length of AD use was 41.4 months (IQR = 24.7-62.8 months). CONCLUSIONS AD use was an independent predictor of meningioma recurrence. This association may be due to mood or affective changes caused by tumor location in CPF regions that may be a sign of early recurrence. The finding calls attention to AD use in the management of patients with meningioma, and warrants further exploration of an underlying relationship.

Is Psychiatric Depression a Presenting Neurologic Sign of Meningioma? A Critical Review of the Literature with Causative Etiology.

BACKGROUND: Benign meningiomas constitute 80%-90% of all meningiomas and represent the most common type of central nervous system tumor in adults. The vast majority of meningiomas are minimally symptomatic or asymptomatic early in their onset and thereby can often result in delayed diagnosis. Early diagnosis of meningioma is critical, as it can maximize treatment options and improve outcomes and survival. Although seizures and focal neurologic deficits are considered to be the most prevalent symptoms, depression also may be an important and significant sign. A subtle neurologic depression may be an even early presenting sign of meningioma and may precede more traditional presenting symptoms. METHODS: We performed a comprehensive literature review that analyzes the results of prospective studies and case reports on this topic. RESULTS: Our findings show evidence to suggest that depression may be correlated with meningioma presentation. Its prevalence is possibly increased with an anterior location of the tumor. CONCLUSIONS: For patients who exhibit nuances of depression without a history of psychiatric illness, an index of suspicion for meningioma may be warranted.

Plasmacytoid dendritic cells induce tolerance predominantly by cargoing antigen to lymph nodes.

Plasmacytoid dendritic cells (pDCs) have been shown to induce tolerance to innocuous antigens. Their migratory properties allow them to take up antigens from the periphery and transport them to the draining lymph nodes or to the thymus. However, pDC-T-cell interaction in the primary and secondary lymphoid organs still remains poorly defined. In this study, we show that resting pDCs loaded with exogenous antigen could induce tolerance when transferred intralymphatically into a single lymph node of wild-type C57BL/6 mice. However, this was a result of antigen transfer from pDCs to endogenous antigen presenting cells and subsequent abortive proliferation of cognate CD4+ T cells. pDCs could not directly induce the proliferation of CD4+ T cells, as observed in mice lacking MHC class II gene. Moreover, pDCs failed to make physical contacts with OT-II cells as revealed by two-photon imaging. Thus, the role of resting pDCs in tolerance induction seems to be independent of its direct interaction with cognate CD4+ T cells.