Adoptive cellular therapy with T cells expressing the dendritic cell growth factor Flt3L drives epitope spreading and antitumor immunity.

Adoptive cell therapies using genetically engineered T cell receptor or chimeric antigen receptor T cells are emerging forms of immunotherapy that redirect T cells to specifically target cancer. However, tumor antigen heterogeneity remains a key challenge limiting their efficacy against solid cancers. Here, we engineered T cells to secrete the dendritic cell (DC) growth factor Fms-like tyrosine kinase 3 ligand (Flt3L). Flt3L-secreting T cells expanded intratumoral conventional type 1 DCs and substantially increased host DC and T cell activation when combined with immune agonists poly (I:C) and anti-4-1BB. Importantly, combination therapy led to enhanced inhibition of tumor growth and the induction of epitope spreading towards antigens beyond those recognized by adoptively transferred T cells in solid tumor models of T cell receptor and chimeric antigen receptor T cell therapy. Our data suggest that augmenting endogenous DCs is a promising strategy to overcome the clinical problem of antigen-negative tumor escape following adoptive cell therapy.

Challenges in evaluating pelvic floor physiotherapy based strategies in low anterior resection syndrome: a systematic review and qualitative analysis.

AIM: Physiotherapy is an established treatment strategy for low anterior resection syndrome (LARS). However, data on its efficacy are limited. This is in part due to the inherent challenges in study design in this context. This systematic review aims to analyse the methodology of studies using pelvic floor physiotherapy for treatment of LARS to elucidate the challenges and limitations faced, which may inform the design of future prospective trials. METHODOLOGY: A systematic review of the literature was undertaken through MEDLINE, Embase and Cochrane Library, yielding 345 unique records for screening. Five studies were identified for review. Content thematic analysis of study limitations was carried out using the Braun and Clarke method. Line-by-line coding was used to organize implicit and explicit challenges and limitations under broad organizing categories. RESULTS: Key challenges fell into five overarching categories: patient-related issues, cancer-related issues, adequate symptomatic control, intervention-related issues and measurement of outcomes. Adherence, attrition and randomization contributed to potential bias within these studies, with imbalance in the baseline patient characteristics, particularly gender and baseline pelvic floor function scores. Outcome measurements consisted of patient-reported measures and quality of life measures, where significant improvements in bowel function according to patient-reported outcome measures were not reflected in the quality of life scores. CONCLUSION: Upcoming trial design in the area of pelvic floor physiotherapy for faecal incontinence related to rectal cancer surgery can be cognisant of and design around the challenges identified in this systematic review, including the reduction of bias, exclusion of the placebo effect and the potential cultural differences in attitude towards a sensitive intervention.

Redundancy in the International Anorectal Physiology Working Group Manometry Protocol: A Diagnostic Accuracy Study in Fecal Incontinence.

BACKGROUND: Anorectal manometry (ARM) is essential for identifying sphincteric dysfunction. The International Anorectal Physiology Working Group (IAPWG) protocol and London Classification provide a standardized format for performing and interpreting ARM. However, there is scant evidence to support timing and number of constituent maneuvers. AIMS: To assess the impact of protocol modification on diagnostic accuracy in patients with fecal incontinence. METHODS: Retrospective analysis of high-resolution ARM recordings from consecutive patients based on the current IAPWG protocol and modifications thereof: (1) baseline rest period (60 vs. 30 vs. 10 s); (2) number of abnormal short squeezes (SS) out of 3 (SS1/SS2/SS3) based on maximal incremental squeeze pressures over 5 s; (3) resting anal pressures (reflecting recovery) at 25-30 versus 15-20 s after SS1. RESULTS: One hundred patients (86 F, median age 55 [IQR: 39-65]; median St. Mark's incontinence score 14 [10-17]) were studied. 26% and 8% had anal hypotonia and hypertonia, respectively. Compared with 60-s resting pressure, measurements had perfect correlation (κ = 1.0) over 30 s, and substantial correlation (κ = 0.85) over 10 s. After SS1, SS2, and SS3, 43%, 49%, and 46% had anal hypocontractility, respectively. Correlation was substantial between SS1 and SS2 (κ = 0.799) and almost perfect between SS2 and SS3 (κ = 0.9). Compared to resting pressure of 5 s before SS1, pressure recordings at 25-30 and 15-20 s after SS1 were significantly correlated. CONCLUSIONS: A 30-s resting anal pressure, analysis of 2 short-squeezes with a 20-s between-maneuver recovery optimizes study duration without compromising diagnostic accuracy. These findings indicate the IAPWG protocol has redundancy.

The development of a faecal incontinence core outcome set: an international Delphi study protocol.

PURPOSE: Faecal incontinence (FI) is estimated to affect around 7.7% of people. There is a lack of uniformity in outcome definitions, measurement and reporting in FI studies. Until now, there is no general consensus on which outcomes should be assessed and reported in FI research. This complicates comparison between studies and evidence synthesis, potentially leading to recommendations not evidence-based enough to guide physicians in selecting an FI therapy. A solution for this lack of uniformity in reporting of outcomes is the development of a Core Outcome Set (COS) for FI. This paper describes the protocol for the development of a European COS for FI. METHODS: Patient interviews and a systematic review of the literature will be performed to identify patient-, physician- and researcher-oriented outcomes. The outcomes will be categorised using the COMET taxonomy and put forward to a group of patients, physicians (i.e. colorectal surgeons, gastroenterologists and general practitioners) and researchers in a Delphi consensus exercise. This exercise will consist of up to three web-based rounds in which participants will prioritise and condense the list of outcomes, which is expected to result in consensus. A consensus meeting with participants from all stakeholder groups will take place to reach a final agreement on the COS. DISCUSSION: This study protocol describes the development of a European COS to improve reliability and consistency of outcome reporting in FI studies, thereby improving evidence synthesis and patient care. TRIAL REGISTRATION: This project has been registered in the COMET database on the 1st of April 2020, available at http://www.comet-initiative.org/Studies/Details/1554 . The systematic review has been registered on the PROSPERO database on the 31st of August 2020, available at https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=202020&VersionID=1381336 .

The London Classification: Improving Characterization and Classification of Anorectal Function with Anorectal Manometry.

PURPOSE OF REVIEW: Objective measurement of anorectal sensorimotor function is a requisite component in the clinical evaluation of patients with intractable symptoms of anorectal dysfunction. Regrettably, the utility of the most established and widely employed investigations for such measurement (anorectal manometry (ARM), rectal sensory testing and the balloon expulsion test) has been limited by wide variations in clinical practice. RECENT FINDINGS: This article summarizes the recently published International Anorectal Physiology Working Group (IAPWG) consensus and London Classification of anorectal disorders, together with relevant allied literature, to provide guidance on the indications for, equipment, protocol, measurement definitions and results interpretation for ARM, rectal sensory testing and the balloon expulsion test. The London Classification is a standardized method and nomenclature for description of alterations in anorectal motor and sensory function using office-based investigations, adoption of which should bring much needed harmonization of practice.

Proctalgia Syndromes: Update in Diagnosis and Management.

PURPOSE OF REVIEW: Functional anorectal pain syndromes are a neglected yet often disabling clinical entity resulting in significant economic and psychological burden to the patient. The aim of this review is to update the practicing gastroenterologist/coloproctologist on the diagnosis and management of these complicated disorders. RECENT FINDINGS: The updated Rome foundation diagnostic criteria (Rome IV) for functional anorectal pain subgroups chronic proctalgia (levator ani syndrome and unspecified functional anorectal pain) and acute proctalgia (proctalgia fugax) on the basis of symptom duration and digital rectal examination findings. Chronic proctalgia is thought to be secondary to paradoxical pelvic floor contraction in many patients and biofeedback to improve the defecation effort has proven effective for over 90% in the short term. Unfortunately, management of proctalgia fugax remains challenging and treatment outcomes modest at best. A number of therapies to relax the pelvic floor may be employed to improve symptoms in functional anorectal pain syndromes; however, only biofeedback to improve defaecatory dynamics in patients with levator ani syndrome has proven effectiveness in a randomized setting. Further investigation of treatment approaches in proctalgia fugax is required.

High-resolution Anorectal Manometry Measures Are More Accurate Than Conventional Measures in Detecting Anal Hypocontractility in Women With Fecal Incontinence.

BACKGROUND & AIMS: Anorectal manometry is a common test of anal tone and contractility. However, existing measures are often criticized for their poor ability to detect functional differences between individuals with presumably normal sphincter function and individuals with fecal incontinence. We investigated whether new measures, derived from high-resolution anal manometry (HR-ARM), more accurately identify incontinent women with abnormal sphincter function than measures from conventional anal manometry (C-ARM). METHODS: We performed a prospective HR-ARM study of 85 healthy female volunteers (median age 41 years; range 18-68 years) and 403 women with fecal incontinence (median age 55; range 18-91 years) from June 2013 through December 2015. We compared the diagnostic accuracy of conventional measurements of contractility (C-ARM squeeze increment) and tone (C-ARM resting pressure) (the reference standards), with HR-ARM measures (HR-ARM squeeze increment, HR-ARM contractile integral, HR-ARM resting average, HR-ARM rest integral). We also investigated associations between symptom severity and structural sphincter integrity. RESULTS: We identified anal hypocontractility in 129 women with fecal incontinence using the C-ARM squeeze increment (32% sensitivity; likelihood ratio, 6.4), in 189 women using the HR-ARM squeeze increment (47% sensitivity; likelihood ratio, 9.4), and in 220 women using the HR-ARM contractile integral (55% sensitivity; likelihood ratio, 11.0). In contrast, HR-ARM measures of anal tone did not particularly outperform conventional measures (31% sensitivity for HR-ARM resting average vs 28% for C-ARM resting pressure). Ninety-eight of the 206 patients with fecal incontinence who were classified as having normal anal function, based on C-ARM measures, were reclassified as having abnormal function based on HR-ARM measures. CONCLUSION: In a prospective study of healthy women and women with fecal incontinence, we demonstrated that the newly developed HR-ARM contractile integral increased the sensitivity of detection of anal hypocontractility, from 32% to 55%, compared with conventional measurements of squeeze.

Selected Toll-like receptor ligands and viruses promote helper-independent cytotoxic T cell priming by upregulating CD40L on dendritic cells.

CD40L (CD154) on CD4(+) T cells has been shown to license dendritic cells (DCs) via CD40 to prime cytotoxic T lymphocyte (CTL) responses. We found that the converse (CD40L on DCs) was also important. Anti-CD40L treatment decreased endogenous CTL responses to both ovalbumin and influenza infection even in the absence of CD4(+) T cells. DCs expressed CD40L upon stimulation with agonists to Toll-like receptor 3 (TLR3) and TLR9. Moreover, influenza infection, which stimulates CTLs without help, upregulated CD40L on DCs, but herpes simplex infection, which elicits CTLs through help, did not. CD40L-deficient (Cd40lg(-/-)) DCs are suboptimal both in vivo in bone marrow chimera experiments and in vitro in mixed lymphocyte reactions. In contrast, Cd40lg(-/-) CD8(+) T cells killed as effectively as wild-type cells. Thus, CD40L upregulation on DCs promoted optimal priming of CD8(+) T cells without CD4(+) T cells, providing a mechanism by which pathogens may elicit helper-independent CTL immunity.

Prosurvival Bcl-2 family members reveal a distinct apoptotic identity between conventional and plasmacytoid dendritic cells.

Dendritic cells (DCs) are heterogeneous, comprising subsets with functional specializations that play distinct roles in immunity as well as immunopathology. We investigated the molecular control of cell survival of two main DC subsets: plasmacytoid DCs (pDCs) and conventional DCs (cDCs) and their dependence on individual antiapoptotic BCL-2 family members. Compared with cDCs, pDCs had higher expression of BCL-2, lower A1, and similar levels of MCL-1 and BCL-XL. Transgenic overexpression of BCL-2 increased the pDC pool size in vivo with only minor impact on cDCs. With a view to immune intervention, we tested BCL-2 inhibitors and found that ABT-199 (the BCL-2 specific inhibitor) selectively killed pDCs but not cDCs. Conversely, genetic knockdown of A1 profoundly reduced the proportion of cDCs but not pDCs. We also found that conditional ablation of MCL-1 significantly reduced the size of both DC populations in mice and impeded DC-mediated immune responses. Thus, we revealed that the two DC types have different cell survival requirements. The molecular basis of survival of different DC subsets thus advocates the antagonism of selective BCL-2 family members for treating diseases pertaining to distinct DC subsets.

The life and death of immune cell types: the role of BCL-2 anti-apoptotic molecules.

Targeting survival mechanisms of immune cells may provide an avenue for immune intervention to dampen unwanted responses (e.g. autoimmunity, immunopathology and transplant rejection) or enhance beneficial ones (e.g. immune deficiency, microbial defence and cancer immunotherapy). The selective survival mechanisms of the various immune cell types also avails the possibility of specific tailoring of such interventions. Here, we review the role of the BCL-2 anti-apoptotic family members (BCL-2, BCL-XL, BCL-W, MCL-1 and A1) on cell death/survival of the major immune cell types, for example, T, NK, B, dendritic cell (DC) lineages. There is both selectivity and redundancy among this family. Selectivity comes partly from the expression levels in each of the cell types. For example, plasmacytoid DC express abundant BCL-2 and are susceptible to BCL-2 antagonism or deficiency, whereas conventional DC express abundant A1 and are susceptible to A1 deficiency. There is, however, also functional redundancy; for example, overexpression of MCL-1 can override BCL-2 antagonism in plasmacytoid DC. Moreover, susceptibility to another anti-apoptotic family member can be unmasked, when one or other member is removed. These dual principles of selectivity and redundancy should guide the use of antagonists for manipulating immune cells.

Diagnostic accuracy study of anorectal manometry for diagnosis of dyssynergic defecation.

OBJECTIVE: The diagnostic accuracy of anorectal manometry (AM), which is necessary to diagnose functional defecatory disorders (FDD), is unknown. Using blinded analysis and standardised reporting of diagnostic accuracy, we evaluated whether AM could discriminate between asymptomatic controls and patients with functional constipation (FC). DESIGN: Derived line plots of anorectal pressure profiles during simulated defecation were independently analysed in random order by three expert observers blinded to health status in 85 women with FC and 85 age-matched asymptomatic healthy volunteers (HV). Using accepted criteria, these pressure profiles were characterised as normal (ie, increased rectal pressure coordinated with anal relaxation) or types I-IV dyssynergia. Interobserver agreement and diagnostic accuracy were determined. RESULTS: Blinded consensus-based assessment disclosed a normal pattern in 16/170 (9%) of all participants and only 11/85 (13%) HV. The combined frequency of dyssynergic patterns (I-IV) was very similar in FC (80/85 (94%)) and HV (74/85 (87%)). Type I dyssynergia ('paradoxical' contraction) was less prevalent in FC (17/85 (20%) than in HV (31/85 (36.5%), p=0.03). After statistical correction, only type IV dyssynergia was moderately useful for discriminating between FC (39/85 (46%)) and HV (17/85 (20%)) (p=0.001, positive predictive value=70.0%, positive likelihood ratio=2.3). Interobserver agreement was substantial or moderate for identifying a normal pattern, dyssynergia types I and IV, and FDD, and fair for types II and III. CONCLUSIONS: While the interpretation of AM patterns is reproducible, nearly 90% of HV have a pattern that is currently regarded as 'abnormal' by AM. Hence, AM is of limited utility for distinguishing between FC and HV.

Colonic and anorectal motility testing in the high-resolution era.

PURPOSE OF REVIEW: The past few years have seen an increase in the number of research and clinical groups around the world using high-resolution manometry (HRM) to record contractile activity in the anorectum and colon. Yet despite the uptake and growing number of publications, the clinical utility and potential advantages over traditional manometry remain undetermined. RECENT FINDINGS: Nearly all of the publications in the field of anorectal and colonic HRM have been published within the last 3 years. These studies have included some data on normal ranges in healthy adults, and abnormalities in patient groups with constipation or fecal incontinence, anal fissure, perineal descent, rectal cancer, and Hirschsprung's disease. Most of the studies have been conducted on adults, with only three published studies in pediatric populations. Very few studies have attempted to show advantages of HRM over traditional manometry SUMMARY: High-resolution anorectal and colonic manometry provide a more comprehensive characterization of motility patterns and coordinated activity; this may help to improve our understanding of the normal physiology and pathophysiology in these regions. To date, however, no published study has conclusively demonstrated a clinical, diagnostic, or interventional advantage over conventional manometry.

The inflammatory cytokine, GM-CSF, alters the developmental outcome of murine dendritic cells.

Fms-like tyrosine kinase 3 ligand (Flt3L) is a major cytokine that drives development of dendritic cells (DCs) under steady state, whereas GM-CSF becomes a prominent influence on differentiation during inflammation. The influence GM-CSF exerts on Flt3L-induced DC development has not been thoroughly examined. Here, we report that GM-CSF alters Flt3L-induced DC development. When BM cells were cultured with both Flt3L and GM-CSF, few CD8⁺ equivalent DCs or plasmacytoid DCs developed compared to cultures supplemented with Flt3L alone. The disappearance of these two cell subsets in GM-CSF + Flt3L culture was not a result of simple inhibition of their development, but a diversion of the original differentiation trajectory to form a new cell population. As a consequence, both DC progeny and their functions were altered. The effect of GM-CSF on DC subset development was confirmed in vivo. First, the CD8⁺ DC numbers were increased under GM-CSF deficiency (when either GM-CSF or its receptor was ablated). Second, this population was decreased under GM-CSF hyperexpression (by transgenesis or by Listeria infection). Our finding that GM-CSF dominantly changes the regulation of DC development in vitro and in vivo has important implications for inflammatory diseases or GM-CSF therapy.

Unlike CD4+ T-cell help, CD28 costimulation is necessary for effective primary CD8+ T-cell influenza-specific immunity.

The importance of costimulation on CD4(+) T cells has been well documented. However, primary CTLs against many infections including influenza can be generated in the absence of CD4(+) T-cell help. The role of costimulation under such "helpless" circumstances is not fully elucidated. Here, we investigated such a role for CD28 using CTLA4Ig transgenic (Tg) mice. To ensure valid comparison across the genotypes, we showed that all mice had similar naïve precursor frequencies and similar peak viral loads. In the absence of help, viral clearance was significantly reduced in CTLA4Ig Tg mice compared with WT mice. CD44(+) BrdU(+) influenza-specific CD8(+) T cells were diminished in CTLA4Ig Tg mice at days 5 and 8 postinfection. Adoptive transfer of ovalbumin-specific transgenic CD8(+) T cells (OT-I)-I cells into WT or CTLA4Ig Tg mice revealed that loss of CD28 costimulation resulted in impairment in OT-I cell division. As shown previously, neither viral clearance nor the generation of influenza-specific CD8(+) T cells was affected by the absence of CD4(+) T cells alone. In contrast, both were markedly impaired by CD28 blockade of "helpless" CD8(+) T cells. We suggest that direct CD28 costimulation of CD8(+) T cells is more critical in their priming during primary influenza infection than previously appreciated.

Defects in the Bcl-2-regulated apoptotic pathway lead to preferential increase of CD25 low Foxp3+ anergic CD4+ T cells.

Defects in the Bcl-2-regulated apoptotic pathway inhibit the deletion of self-reactive T cells. What is unresolved, however, is the nature and fate of such self-reactive T cells escaping deletion. In this study, we report that mice with such defects contained increased numbers of CD25(low)Foxp3(+) cells in the thymus and peripheral lymph tissues. The increased CD25(low)Foxp3(+) population contained a large fraction of cells bearing self-reactive TCRs, evident from a prominent increase in self-superantigen-specific Foxp3(+)Vß5(+)CD4(+) T cells in BALB/c Bim(-/-) mice compared with control animals. The survival rate of the expanded CD25(low)Foxp3(+) cells was similar to that of CD25(high)Foxp3(+) CD4 T cells in vitro and in vivo. IL-2R stimulation, but not TCR ligation, upregulated CD25 on CD25(low)Foxp3(+)CD4(+) T cells in vitro and in vivo. The expanded CD25(low)Foxp3(+)CD4(+) T cells from Bim(-/-) mice were anergic but also had weaker regulatory function than CD25(high)Foxp3(+) CD4(+) T cells from the same mice. Analysis of Bim(-/-) mice that also lacked Fas showed that the peripheral homeostasis of this expanded population was in part regulated by this death receptor. In conclusion, these results show that self-reactive T cell escapes from thymic deletion in mice defective in the Bcl-2-regulated apoptotic pathway upregulate Foxp3 and become unresponsive upon encountering self-Ag without necessarily gaining potent regulatory function. This clonal functional diversion may help to curtail autoaggressiveness of escaped self-reactive CD4(+) T cells and thereby safeguard immunological tolerance.

TNF receptor 1 deficiency increases regulatory T cell function in nonobese diabetic mice.

TNF has been implicated in the pathogenesis of type 1 diabetes. When administered early in life, TNF accelerates and increases diabetes in NOD mice. However, when administered late, TNF decreases diabetes incidence and delays onset. TNFR1-deficient NOD mice were fully protected from diabetes and only showed mild peri-insulitis. To further dissect how TNFR1 deficiency affects type 1 diabetes, these mice were crossed to ß cell-specific, highly diabetogenic TCR transgenic I-A(g7)-restricted NOD4.1 mice and Kd-restricted NOD8.3 mice. TNFR1-deficient NOD4.1 and NOD8.3 mice were protected from diabetes and had significantly less insulitis compared with wild type NOD4.1 and NOD8.3 controls. Diabetic NOD4.1 mice rejected TNFR1-deficient islet grafts as efficiently as control islets, confirming that TNFR1 signaling is not directly required for ß cell destruction. Flow cytometric analysis showed a significant increase in the number of CD4(+)CD25(+)Foxp3(+) T regulatory cells in TNFR1-deficient mice. TNFR1-deficient T regulatory cells were functionally better at suppressing effector cells than were wild type T regulatory cells both in vitro and in vivo. This study suggests that blocking TNF signaling may be beneficial in increasing the function of T regulatory cells and suppression of type 1 diabetes.

BH3 mimetics antagonizing restricted prosurvival Bcl-2 proteins represent another class of selective immune modulatory drugs.

Death by apoptosis shapes tissue homeostasis. Apoptotic mechanisms are so universal that harnessing them for tailored immune intervention would seem challenging; however, the range and different expression levels of pro- and anti-apoptotic molecules among tissues offer hope that targeting only a subset of such molecules may be therapeutically useful. We examined the effects of the drug ABT-737, a mimetic of the killer BH3 domain of the Bcl-2 family of proteins that induces apoptosis by antagonizing Bcl-2, Bcl-X(L), and Bcl-W (but not Mcl-1 and A1), on the mouse immune system. Treatment with ABT-737 reduced the numbers of selected lymphocyte and dendritic cell subpopulations, most markedly in lymph nodes. It inhibited the persistence of memory B cells, the establishment of newly arising bone marrow plasma cells, and the induction of a cytotoxic T cell response. Preexisting plasma cells and germinal centers were unaffected. Notably, ABT-737 was sufficiently immunomodulatory to allow long-term survival of pancreatic allografts, reversing established diabetes in this model. These results provide an insight into the selective mechanisms of immune cell survival and how this selectivity avails a different strategy for immune modulation.

Case report: Acute appendicitis in appendix duplication.

INTRODUCTION: Duplication of the appendix is a very rare presentation. According to the Cave-Wallbridge classification, there are three types of duplicate appendix. PRESENTATION OF CASE: A 43 year old female presented with classical symptoms of acute appendicitis, with unremarkable inflammatory markers. The diagnosis was confirmed on pre-operative computer tomography (CT). During laparoscopy two tubular structures were identified: one arising from the tenia libera of the caecum adjacent to the terminal ileum and one retrocaecally at the confluence of the teniae. Both structures were excised using a laparoscopic linear stapler. Histopathological analysis demonstrated the accessory structure to be a microscopically unremarkable blind-ended tubular structure. The other specimen demonstrated acute gangrenous inflammation of the appendix. The patient had an uneventful recovery and was discharged home the following day. DISCUSSION: Appendix duplication is rare; however, failure to recognise it in a patient with acute appendicitis could result in a retained source of sepsis, requiring subsequent re-exploration of the abdomen. The case presented here represents a Type B2 according to the Cave-Wallbridge classification and is the most susceptible to inadvertent error due to having appendixes in both typical and atypical anatomical locations. This case also highlights the probability of this diagnosis being missed on pre-operative CT. CONCLUSION: This case report presents a unique opportunity for surgical trainees to review intra-operative laparoscopic images of a duplicate appendix, both to allow them to recognise this pathology if encountered in the future, and to embed the importance of ruling it out with thorough intra-operative examination.

Reducing or increasing ß-cell apoptosis without inflammation does not affect diabetes initiation in neonatal NOD mice.

The presentation of islet antigens in the pancreatic LNs (PLNs) of mice is a developmentally regulated process. It has been hypothesized that, during physiological tissue remodeling, a wave of neonatal ß-cell apoptosis may initiate diabetes in autoimmune-prone strains of mice. If true, increasing or decreasing physiological ß-cell apoptosis in neonatal NOD mice should alter the time-course of antigen presentation in the PLNs. We used transgenic over-expression of either an anti-apoptotic protein (Bcl-2) or a toxic transgene (rat insulin promoter-Kb) in mouse ß cells to reduce or increase neonatal ß-cell apoptosis, respectively. Neither intervention affected the timing of antigen presentation in the PLNs or the initiation of islet infiltration. This suggests that under physiological conditions and in the absence of inflammation, neonatal ß-cell apoptosis in NOD mice is not the trigger for antigen presentation in the draining LNs.

GM-CSF increases cross-presentation and CD103 expression by mouse CD8⁺ spleen dendritic cells.

Resident CD8(+) DCs perform several functions, including cross-presenting antigen and rapidly engulfing the Gram-positive intracellular pathogen Listeria monocytogenes. Little is known about how these functions of CD8(+) DCs are modulated. Here, we show that granulocyte-macrophage CSF (GM-CSF), a cytokine that exists at low levels at steady state but is elevated during infection and inflammation, enhances cross-presentation and rapid uptake of L. monocytogenes by resident CD8(+) DCs. This previously unrecognized functional enhancement of CD8(+) DCs by GM-CSF was independent of promoting DC survival in vitro. Enhancement of these functions by GM-CSF was also marked by CD103 expression on CD8(+) DCs that was strongly regulated by GM-CSF. Our findings not only identify GM-CSF as a key molecule regulating CD8(+) DC function, but also as a factor responsible for functional heterogeneity of CD8(+) DCs that is at least substantially demarcated by CD103 expression.