Pilot Study of a Wearable Activity Monitor During Head and Neck Radiotherapy to Predict Clinical Outcomes.

PURPOSE: Given the established associations between performance status and survival in a variety of cancers, there is significant interest in using a biometric wearable device (WD) to predict outcomes in the oncology population. In this pilot study, we investigated the ability of a WD to predict meaningful clinical end points in patients undergoing head and neck radiotherapy. METHODS: Patients receiving head and neck definitive chemoradiotherapy or postoperative radiotherapy/chemoradiotherapy were enrolled in this pilot study, designed to show 90% compliance with using the device. Individuals were asked to wear the WD for 23 hours a day, and hospital admissions, pain medication usage, and FACT-G quality-of-life (QoL) score were prospectively recorded. RESULTS: Fifty-one patients were enrolled and started using the WD, but eight patients stopped wearing it, resulting in a compliance probability of only 84%. There were 15 hospital admissions, 13 of which were planned for feeding tube placement. There was no step count threshold that predicted the need for admission or more pain medications. However, among the 25 patients with a significant reduction in FACT-G score, the average reductions in daily steps during the week and weekend before the decline were 988 (P = .005) and 1,311 (P = .018), respectively, and the odds of a QoL reduction were more than 4-fold higher among patients experiencing a week-to-week reduction of at least 1,000 daily steps. There was no association between heart rate and any end point. CONCLUSION: Although not meeting the compliance goal, the majority of patients did use the WD. The WD signal could not identify patients requiring hospitalization or significantly more pain medication, but the finding of reduced step counts before a significant reduction in QoL is provocative.

Prospective Phase 2 Study of Radiation Therapy Dose and Volume De-escalation for Elective Neck Treatment of Oropharyngeal and Laryngeal Cancer.

PURPOSE: The required elective nodal dose and volumes for head and neck intensity modulated radiation therapy have largely been extrapolated from conventional radiation therapy fields. In this prospective, dual-center, phase 2 study, we investigated the efficacy and tolerability of reduced elective nodal volume and dose in oropharyngeal and laryngeal squamous cell carcinoma. METHODS AND MATERIALS: Patients with newly diagnosed squamous cell carcinoma of the oropharynx and larynx were eligible for enrollment. Each lymph node was characterized as involved or suspicious based on imaging criteria. For oropharynx cancer, only involved and immediately adjacent stations were treated to 40 Gy in 20 fractions. In larynx patients, at least bilateral levels II and III were treated to 40 Gy, with level IV treated only if level III was involved. Involved and suspicious nodes were then boosted with 30 Gy and 24 Gy in 15 fractions, respectively. Concurrent chemotherapy was required for stage T3N0-1 and IVA/B patients. The primary endpoint of the study was solitary elective volume recurrence, with secondary endpoints including patterns of failure and patient-reported outcomes. RESULTS: A total of 72 (51 oropharynx, 21 larynx) patients completed treatment on this trial from January 2017 through November 2018. The stages at presentations were 5, 17, and 50 stage I-II, III, and IV, respectively, with 90% treated with chemoradiation therapy. At a median follow-up of 24.7 months for surviving patients, there have been no solitary elective nodal recurrences. Seven patients developed a nodal recurrence, 5 of which were in-field and 2 were elective with synchronous in-field recurrence. Patient-reported outcomes assessment at 1 year showed superior or equivalent outcomes compared with baseline, except for saliva and taste measures. CONCLUSIONS: The results of this trial suggest that elective dose and volume reduction is oncologically sound for oropharyngeal and laryngeal cancer treated with intensity modulated radiation therapy, with promising quality-of-life outcomes.

Phase 1 Fractional Dose-Escalation Study of Equipotent Stereotactic Radiation Therapy Regimens for Early-Stage Glottic Larynx Cancer.

PURPOSE: Early-stage glottic larynx squamous cell carcinoma (GLC) is a relatively common disease with excellent oncologic control, but treatment is associated with acute dysphagia and long-term voice quality changes. This phase 1 study of hypofractionated radiation therapy for early-stage GLC increased the fraction size while reducing the number of fractions until 5-fraction stereotactic ablative radiation therapy (SABR) was delivered. METHODS AND MATERIALS: Eligible patients had received a diagnosis of stage Tis to T2 GLC. Patients who had undergone prior curative-intent surgery were excluded. The equipotent dose levels were as follows: (1) level 0, 50 Gy in 15 fractions (n = 4); (2) level 1, 45 Gy in 10 fractions (n = 13); and (3) level 2, 42.5 Gy in 5 fractions (SABR level, n = 12). Grade 3 or 4 laryngeal edema, voice, dyspnea, stridor, or cough were the predefined dose-limiting toxicities. RESULTS: Twenty-nine patients were enrolled from November 2013 to March 2017. The median and minimum follow-up times were 39.2 and 13 months, respectively. Two actively smoking patients, 1 treated in level 1 (grade 4 laryngeal edema, grade 3 dysphagia) and 1 treated in level 2 (grade 3 laryngeal necrosis, dysphagia), developed dose-limiting toxicities. The former patient soon developed a local recurrence, and the latter patient recovered. There were a total of 5 local recurrences: 2 in level 0 and 3 in level 1. The Voice Handicap Index results showed robust long-term voice quality with median values of 7 and 0 at 6 and 12 months, respectively. CONCLUSIONS: Given the tolerability, excellent voice outcomes, and preliminary efficacy data of 5-fraction glottic larynx SABR, this regimen warrants further study.

Combining many-objective radiomics and 3D convolutional neural network through evidential reasoning to predict lymph node metastasis in head and neck cancer.

Lymph node metastasis (LNM) is a significant prognostic factor in patients with head and neck cancer, and the ability to predict it accurately is essential to optimizing treatment. Positron emission tomography (PET) and computed tomography (CT) imaging are routinely used to identify LNM. Although large or highly active lymph nodes (LNs) have a high probability of being positive, identifying small or less reactive LNs is challenging. The accuracy of LNM identification strongly depends on the physician's experience, so an automatic prediction model for LNM based on CT and PET images is warranted to assist LMN identification across care providers and facilities. Radiomics and deep learning are the two promising imaging-based strategies for node malignancy prediction. Radiomics models are built based on handcrafted features, while deep learning learns the features automatically. To build a more reliable model, we proposed a hybrid predictive model that takes advantages of both radiomics and deep learning based strategies. We designed a new many-objective radiomics (MaO-radiomics) model and a 3D convolutional neural network (3D-CNN) that fully utilizes spatial contextual information, and we fused their outputs through an evidential reasoning (ER) approach. We evaluated the performance of the hybrid method for classifying normal, suspicious and involved LNs. The hybrid method achieves an accuracy (ACC) of 0.88 while XmasNet and Radiomics methods achieve 0.81 and 0.75, respectively. The hybrid method provides a more accurate way for predicting LNM using PET and CT.

Comparative effectiveness of primary radiotherapy versus surgery in elderly patients with locally advanced oropharyngeal squamous cell carcinoma.

OBJECTIVES: To determine the comparative effectiveness of primary radiotherapy (RT) and primary surgery (PS) for locally advanced oropharyngeal squamous cell carcinoma (OPSCC). MATERIALS AND METHODS: Eligible individuals were patients in the SEER-Medicare registry diagnosed with locally advanced OPSCC between 2000 and 2011. Patients were categorized as receiving either primary RT ±â€¯chemotherapy, or PS ±â€¯adjuvant RT or chemoradiotherapy (CRT). Overall survival (OS) was analyzed using Cox multivariable analysis (MVA). Risks of gastrostomy dependence (GD), esophageal stricture (ES), and osteoradionecrosis (ORN) were analyzed using logistic regression. RESULTS: A total of 2754 patients (69% RT, 31% PS) were included in this cohort, with a median age of 72 years. Patients treated with RT, CRT and PS experienced 3-year OS outcomes of 36.1%, 52.8%, and 54.9%, respectively (p < 0.001). Increasing age, unmarried status, increasing comorbidity, lower income, base of tongue (BOT) site, higher stage, no prior PET, and RT alone (but not CRT) were associated with inferior OS. Independent predictors of GD at 6 months included black race, BOT site, advanced stage, and CRT. The risks of ORN and stricture were not associated with treatment modality. Concurrent chemotherapy improved OS with definitive RT but had no impact in adjuvant RT. Only cisplatin- and taxane-containing regimens improved OS, but all concurrent agents, including cetuximab, significantly worsened GD. CONCLUSION: Local therapy decisions for locally advanced OPSCC must be individualized, with CRT increasing acute and chronic GD. The differential survival impact of concurrent chemotherapy in the definitive and adjuvant setting may be a consideration in decision-making.

Risk of Unplanned Hospital Encounters in Patients Treated With Radiotherapy for Head and Neck Squamous Cell Carcinoma.

CONTEXT: Radiotherapy is highly effective for treating squamous cell carcinoma of the head and neck but is often associated with significant toxicities and severe morbidity. Unplanned emergency department (ED) visits and hospitalizations are common during treatment and come with a substantial financial and health burden as well as the potential for impaired long-term outcomes due to treatment disruption. OBJECTIVES: The objective of this study was to identify patient, disease, and treatment characteristics that were associated with ED encounters and admissions. METHODS: A cohort of 462 patients with cancer of the head and neck treated with radiotherapy at UT Southwestern between 2010 and 2015 was retrospectively analyzed. The risks of ED visits, admissions, multiple admissions, and extended admissions were determined. Risk factors for an unplanned hospital encounter were analyzed using univariate and multivariate logistic regression. RESULTS: Overall, 36% of patients had an unplanned hospital encounter during the treatment window. Patients with advanced disease, those with high comorbidity score, and those treated with concurrent chemotherapy were more likely to have unplanned admissions/ED visits. Social factors such as marital status, smoking status, and registration in the public hospital system were also strongly associated with admissions and multiple encounters. CONCLUSION: The high rate of admissions and ED visits emphasizes the importance of anticipating and managing toxicities during treatment. Social factors have a strong association with unplanned encounters and may present opportunities for targeted interventions to reduce admissions for patients at highest risk.

Predicting Lymph Node Metastasis in Head and Neck Cancer by Combining Many-objective Radiomics and 3-dimensioal Convolutional Neural Network through Evidential Reasoning.

Lymph node metastasis (LNM) is a significant prognostic factor in patients with head and neck cancer, and the ability to predict it accurately is essential for treatment optimization. PET and CT imaging are routinely used for LNM identification. However, uncertainties of LNM always exist especially for small size or reactive nodes. Radiomics and deep learning are the two preferred imaging-based strategies for node malignancy prediction. Radiomics models are built based on handcrafted features, and deep learning can learn the features automatically. We proposed a hybrid predictive model that combines many-objective radiomics (MO-radiomics) and 3-dimensional convolutional neural network (3D-CNN) through evidential reasoning (ER) approach. To build a more reliable model, we proposed a new many-objective radiomics model. Meanwhile, we designed a 3D-CNN that fully utilizes spatial contextual information. Finally, the outputs were fused through the ER approach. To study the predictability of the two modalities, three models were built for PET, CT, and PET& CT. The results showed that the model performed best when the two modalities were combined. Moreover, we showed that the quantitative results obtained from the hybrid model were better than those obtained from MO-radiomics and 3D-CNN.

Cost-Effectiveness Analysis of Intensity Modulated Radiation Therapy Versus Proton Therapy for Oropharyngeal Squamous Cell Carcinoma.

PURPOSE: To compared the cost-effectiveness of intensity modulated proton beam therapy (PBT) and intensity modulated radiation therapy (IMRT) in the management of stage III-IVB oropharynx cancer (OPC). METHODS AND MATERIALS: A Markov model was constructed to compare IMRT with PBT for a 65-year-old patient with stage IVA OPSCC. We assumed PBT led to a 25% reduction in long-term xerostomia, short-term dysgeusia, and the need for gastrostomy tube. Fewer dental complications were also expected with PBT. Incremental cost-effectiveness ratios (ICERs) were calculated, and value of information analyses were performed. The societal willingness-to-pay was defined as $100K per quality-adjusted life year (QALY). RESULTS: The ICERs for PBT for favorable human papillomavirus (HPV)-positive OPC were $288,000/QALY and $390,000/QALY in the payer perspective (PP) and societal perspective, respectively. Under nearly every scenario, PBT was not cost-effective, with ICERs above $150,000/QALY in the PP. The ICERs for HPV-negative OPC were typically greater than $250K/QALY in both perspectives. For HPV-positive patients, the ICER was less than $100,000/QALY in the PP only in younger patients who experienced a 50% reduction in both xerostomia and gastrostomy use. On probabilistic sensitivity analyses, there were 0% and 0.4% probabilities that PBT was cost-effective for 65- and 55-year old patients, respectively. The value of information was zero or negligible for all ages and perspectives at willingness-to-pay of $100,000/QALY and only meaningful in the PP for younger patients at a willingness-to-pay of $150,000/QALY. CONCLUSIONS: Intensity modulated proton beam therapy was only cost-effective in the PP if assumed to achieve profound reductions in long-term morbidity for younger patients; it was never cost-effective in the societal perspective. Prospective data are needed (and may be valuable) to better characterize the comparative toxicities of these treatments but are unlikely to change this calculation, except potentially in the most favorable cohort of patients.

Risk of contralateral nodal failure following ipsilateral IMRT for node-positive tonsillar cancer.

PURPOSE: To determine the risk of contralateral nodal failure following ipsilateral radiotherapy in a series of patients with node-positive tonsillar squamous cell carcinoma. METHODS: Retrospective review was used to identify 34 patients with well-lateralized node-positive tonsillar squamous cell carcinoma treated with definitive or adjuvant radiation to the primary site and ipsilateral neck between 2005 and 2015. Contralateral nodal failure, locoregional recurrence, distant metastasis, and overall survival were calculated using actuarial and/or cumulative incidence statistics. RESULTS: At last follow-up, contralateral nodal failure was only observed in 1 patient (3%) with N1 disease. At median follow-up of 34 months for surviving patients, the 3-year overall survival probability was 87%, and the 3 year cumulative incidences of locoregional failure and distant metastasis were 6.5% and 7.2%, respectively. No disease-free patient was permanently gastrostomy-dependent. CONCLUSION: Ipsilateral radiation treatment with IMRT is effective in node-positive patients with well-lateralized tonsillar cancer, resulting in a low risk of contralateral regional recurrence, even in patients with N2b disease.

Tim-3 directly enhances CD8 T cell responses to acute Listeria monocytogenes infection.

T cell Ig and mucin domain (Tim) 3 is a surface molecule expressed throughout the immune system that can mediate both stimulatory and inhibitory effects. Previous studies have provided evidence that Tim-3 functions to enforce CD8 T cell exhaustion, a dysfunctional state associated with chronic stimulation. In contrast, the role of Tim-3 in the regulation of CD8 T cell responses to acute and transient stimulation remains undefined. To address this knowledge gap, we examined how Tim-3 affects CD8 T cell responses to acute Listeria monocytogenes infection. Analysis of wild-type (WT) mice infected with L. monocytogenes revealed that Tim-3 was transiently expressed by activated CD8 T cells and was associated primarily with acquisition of an effector phenotype. Comparison of responses to L. monocytogenes by WT and Tim-3 knockout (KO) mice showed that the absence of Tim-3 significantly reduced the magnitudes of both primary and secondary CD8 T cell responses, which correlated with decreased IFN-γ production and degranulation by Tim-3 KO cells stimulated with peptide Ag ex vivo. To address the T cell-intrinsic role of Tim-3, we analyzed responses to L. monocytogenes infection by WT and Tim-3 KO TCR-transgenic CD8 T cells following adoptive transfer into a shared WT host. In this setting, the accumulation of CD8 T cells and the generation of cytokine-producing cells were significantly reduced by the lack of Tim-3, demonstrating that this molecule has a direct effect on CD8 T cell function. Combined, our results suggest that Tim-3 can mediate a stimulatory effect on CD8 T cell responses to an acute infection.

Epitope specificity of memory CD8+ T cells dictates vaccination-induced mortality in LCMV-infected perforin-deficient mice.

Perforin-deficient (PKO) mice serve as models for familial hemophagocytic lympho-histiocytosis, a uniformly fatal disease associated with viral infection of perforin-deficient humans. Naïve perforin-deficient BALB/c mice survive while vaccinated PKO mice containing virus-specific memory CD8(+) T cells rapidly succumb to lymphocytic choriomeningitis virus (LCMV) infection. Thus, vaccination converts a nonlethal persistent infection into a fatal disease mediated by virus-specific memory CD8(+) T cells. Here, we determine the extent to which vaccination-induced mortality in PKO mice following LCMV challenge is due to differences in vaccine modalities, the quantity or epitope specificity of memory CD8(+) T cells. We show that LCMV-induced mortality in immune PKO mice is independent of vaccine modalities and that the starting number of memory CD8(+) T cells specific to the immunodominant epitope NP(118-126) dictates the magnitude of secondary CD8(+) T-cell expansion, the inability to regulate production of CD8(+) T-cell-derived IFN-γ, and mortality in the vaccinated PKO mice. Importantly, mortality is determined by the epitope specificity of memory CD8(+) T cells and the associated degree of functional exhaustion and cytokine dysregulation but not the absolute magnitude of CD8(+) T-cell expansion. These data suggest that deeper understanding of the parameters that influence the outcome of vaccine-induced diseases would aid rational vaccine design to minimize adverse outcomes after infection.

NFIL3/E4BP4 is a key transcription factor for CD8α⁺ dendritic cell development.

Antigen presentation by mature dendritic cells (DCs) is the first step for initiating adaptive immune responses. DCs are composed of heterogeneous functional subsets; however, the molecular mechanisms that regulate differentiation of specific DC subsets are not understood. Here, we report that the basic leucine zipper transcription factor NFIL3/E4BP4 is essential for the development of CD8α(+) conventional DCs (cDCs). Nfil3(-/-) mice specifically lack CD8α(+) cDCs but not CD8α(-) cDCs or plasmacytoid DCs in lymphoid tissues. Flt3 ligand-dependent generation of CD8α(+) cDCs in lymphoid tissues and CD8α(+)-equivalent cDCs from Nfil3(-/-) bone marrow cells was also impaired. NFIL3 regulates CD8α(+) cDC development in part through Batf3 expression. Importantly, Nfil3(-/-) mice exhibited impaired cross-priming of CD8(+) T cells against cell-associated antigen, a process normally performed by CD8α(+) cDCs, and failed to produce IL-12 after TLR3 stimulation. Thus, NFIL3 plays an essential role in the development of CD8α(+) cDCs.

Differential role of "Signal 3" inflammatory cytokines in regulating CD8 T cell expansion and differentiation in vivo.

Following an infection, naïve CD8 T cells are stimulated by dendritic cells (DC) displaying pathogen-derived peptides on MHC class I molecules (signal 1) and costimulatory molecules (signal 2). Additionally, pathogen-induced inflammatory cytokines also act directly on the responding CD8 T cells to regulate their expansion and differentiation. In particular, both type I interferons (IFNs) and IL-12 have been described as critical survival signals (signal 3) for optimal CD8 T cell accumulation during the expansion phase. Furthermore, expansion in numbers of antigen-specific CD8 T cells is coupled with their acquisition of effector functions to combat the infection. However, it still remains unclear whether these same cytokines also regulate the effector/memory differentiation program of the CD8 T cell response in vivo. Here, we demonstrate that defective signaling by either type I IFNs or IL-12 to the responding CD8 T cells impairs maximal expansion in response to DC immunization + CpG ODN, but neither of these cytokines is essential to regulate the effector/memory differentiation program. In addition, lack of direct IL-12 signaling to CD8 T cells accelerates the development of central memory phenotype in both primary and secondary antigen-specific memory CD8 T cells.

Exploiting cross-priming to generate protective CD8 T-cell immunity rapidly.

The number of memory CD8 T cells generated by infection or vaccination correlates strongly with the degree of protection observed in infection and tumor models. Therefore, rapid induction of protective numbers of effector and memory CD8 T cells may be crucial in the case of malignancy, pandemic infection, or bioterrorism. Many studies have shown that amplifying T-cell numbers by prime-boost vaccination is most effective with a substantial time interval between immunizations. In contrast, immunization with peptide-coated mature dendritic cells (DCs) results in a CD8 T-cell response exhibiting accelerated acquisition of memory characteristics, including the ability to respond to booster immunization within days of initial priming. However, personalized DC immunization is too costly, labor intensive, and time-consuming for large-scale vaccination. Here, we demonstrate that in vivo cross-priming with cell-associated antigens or antigen-coated, biodegradable microspheres in the absence of adjuvant quickly generates CD8 T cells that display the phenotype and function of long-term memory populations. Importantly, cross-primed CD8 T cells can respond to booster immunization within days of the initial immunization to generate rapidly large numbers of effector and memory T cells that can protect against bacterial, viral, and parasitic infections, including lethal influenza and malaria-causing Plasmodium infection. Thus, accelerated CD8 T-cell memory after in vivo cross-priming in the absence of adjuvant is generalizable and can be exploited to generate protective immunity rapidly.

T cell epitope specificity and pathogenesis of mouse hepatitis virus-1-induced disease in susceptible and resistant hosts.

Intranasal mouse hepatitis virus-1 (MHV-1) infection of susceptible mouse strains mimics some important pathologic features observed in the lungs of severe acute respiratory syndrome (SARS)-coronavirus-infected humans. The pathogenesis of SARS remains poorly understood, although increasing evidence suggests that immunopathology could play an important role. We previously reported that the adaptive immune response plays an important protective role in MHV-1-infected resistant B6 mice and that both CD4 and CD8 T cells play a significant role in the development of morbidity and lung pathology following intranasal MHV-1 infection of susceptible C3H/HeJ and A/J mice. In this study, we have identified novel CD4 and CD8 epitopes in MHV-1-infected susceptible and resistant strains of mice. Susceptible C3H/HeJ mice mount robust and broad MHV-1-specific CD4 T cell responses, whereas in resistant B6 mice, Ag-specific CD8 T cell responses dominate. We also show that previously immunized susceptible C3H/HeJ mice do not develop any morbidity and are completely protected following a lethal-dose MHV-1 challenge despite mounting only a modest secondary T cell response. Finally, we demonstrate that the resistance displayed by B6 mice is not solely accounted for by the elaboration of a broad and vigorous MHV-1-specific CD8 T cell response, as MHV-1 infection of C3.SW-H2(b)/SnJ mice, which mount an equally robust CD8 T cell response of the same specificity, is still associated with significant morbidity. Thus, identification of novel CD4 and CD8 T cell epitopes for MHV-1 permitted high-resolution analyses of pulmonary T cell responses in a mouse model of SARS.

Tracking the total CD8 T cell response to infection reveals substantial discordance in magnitude and kinetics between inbred and outbred hosts.

Determining the magnitude and kinetics, together with the phenotypic and functional characteristics of responding CD8 T cells, is critical for understanding the regulation of adaptive immunity as well as in evaluating vaccine candidates. Recent technical advances have allowed tracking of some CD8 T cells responding to infection, and a body of information now exists describing phenotypic changes that occur in CD8 T cells of known Ag-specificity during their activation, expansion, and memory generation in inbred mice. In this study, we demonstrate that Ag but not inflammation-driven changes in expression of CD11a and CD8alpha can be used to distinguish naive from Ag-experienced (effector and memory) CD8 T cells after infection or vaccination. Interestingly and in contrast to inbred mice, tracking polyclonal CD8 T cell responses with this approach after bacterial and viral infections revealed substantial discordance in the magnitude and kinetics of CD8 T cell responses in outbred hosts. These data reveal limitations to the use of inbred mouse strains as preclinical models at vaccine development and suggest the same dose of infection or vaccination can lead to substantial differences in the magnitude and timing of Ag-specific CD8 expansion as well in differences in protective memory CD8 T cell numbers in outbred individuals. This concept has direct relevance to development of vaccines in outbred humans.

A default pathway of memory CD8 T cell differentiation after dendritic cell immunization is deflected by encounter with inflammatory cytokines during antigen-driven proliferation.

Inflammatory cytokines induced by infection or vaccination with adjuvant act directly or indirectly on CD8 T cells to modulate their expansion, contraction, and acquisition of memory characteristics. Importantly, the initial exposure of naive T cells to inflammatory cytokines may occur before, during, or after their interaction with stimulating dendritic cells (DC) and it is unknown whether and how the timing of cytokine exposure impacts the CD8 T cell response. In this study, we use an immunization strategy with peptide-coated mature DC that, in the absence of inflammatory cytokines, results in a transient effector phase followed by the accelerated acquisition of memory characteristics by the responding CD8 T cells. Induction of inflammatory cytokines by TLR agonists, at the time of DC immunization or 2-4 days after DC immunization, prevented the early acquisition of memory characteristics by the responding CD8 T cells. Interestingly, although induction of inflammatory cytokines at the time of DC immunization increased the effector response, induction of inflammatory cytokines after DC immunization did not promote further expansion of the responding CD8 T cells but still prevented their early acquisition of memory characteristics. In contrast, induction of inflammatory cytokines 2 days before DC immunization did not prevent the CD8 T cells from early acquisition of memory characteristics. Furthermore, TLR ligand-induced inflammatory cytokines had the most significant impact on the phenotype and function of proliferating CD8 T cells. These data suggest that a default pathway of memory CD8 T cell differentiation is deflected toward sustained effector commitment by encounter with inflammatory cytokines during Ag-driven proliferation.

High initial frequency of TCR-transgenic CD8 T cells alters inflammation and pathogen clearance without affecting memory T cell function.

In adoptive transfer experiments, the initial frequency of naïve TCR-transgenic T cells impacts CD8 T cell phenotype after acute infections. The exact reasons for the observed changes, however, are unclear and it is unknown whether alterations in phenotype translate into impaired memory T cell function as well. Here we perform in vivo comparisons of effector and memory CD8 T cells generated from high or low numbers of naïve precursors. We show that high numbers of adoptively transferred T cells exhibit effector functions that alter systemic inflammation and pathogen abundance in the initial days after infection. While these altered environmental conditions resulted in profound changes in primary effector and memory CD8 T cell phenotype, memory T cells derived from both high and low numbers of naïve precursors protected equally well against re-infection and generated secondary effector and memory T cells that were similar in numbers and phenotype. Our results confirm the necessity to use low numbers of naïve precursors to mimic endogenous immune responses but show at the same time that memory CD8 T cell function in adoptive transfers is independent of input numbers.

NF1 regulatory element functions as a repressor of tissue plasminogen activator expression.

OBJECTIVE: Analysis of the distribution of endothelial cell tissue plasminogen activator (tPA) in the vasculature of rodents and primates demonstrated that tPA is constitutively expressed predominantly in small artery endothelial cells of brain and lung. The regulatory elements responsible for the highly selective expression of arterial endothelial cell tissue plasminogen activator were sought. METHODS AND RESULTS: Transcription factor binding sites were defined by electrophoretic mobility-shift assay (EMSA) analysis using rat lung and brain nuclear extracts and the tPA promoter sequence from -609 to +37 bp. Protein binding to the promoter was found to be mediated by an NF1 site between -158 and -145 bp upstream from the transcriptional start site. Specific binding was confirmed through mutational analysis and competition binding studies. Infection of endothelial cells with a tPA promoter-green fluorescent protein (GFP) (-609 to +37 bp) reporter construct resulted in expression of the GFP, whereas no expression was found in smooth muscle cells. Mutation of the NF1 site increased the GFP expression indicating that the element acts as a repressor. CONCLUSIONS: These results suggest that the 600 bp of the tPA promoter upstream of the transcription start site conveys cell specificity to tPA expression and that an NF1 site within this region acts as a repressor.