High Numbers Of Dendritic Cells Research Articles

Bio-adhesive Macroporous Hydrogels for In Situ Recruitment and Modulation of Dendritic Cells.

Biomaterials that enable in situ recruitment and modulation of immune cells have demonstrated tremendous promise for developing potent cancer immunotherapy such as therapeutic cancer vaccine. One challenge related to biomaterial scaffold-based cancer vaccines is the development of macroporous materials that are biocompatible and stable, enable controlled release of chemokines to actively recruit a large number of dendritic cells (DCs), contain macropores that are large enough to home the recruited DCs, and support the survival and proliferation of DCs. Bio-adhesive macroporous gelatin hydrogels were synthesized and characterized for mechanical properties, porous structure, and adhesion towards tissues. The recruitment of immune cells including DCs to chemokine-loaded bioadhesive macroporous gels was analyzed. The ability of gels loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor extracellular vesicles (EVs) to elicit tumor-specific CD8+ T cell responses was also analyzed. Here we develop a bioadhesive macroporous hydrogel that can strongly adhere to tissues, contain macropores that are large enough to home immune cells, are mechanically tough, and enable controlled release of chemokines to recruit and modulate immune cells in situ. The macroporous hydrogel is composed of a double crosslinked network of gelatin and polyacrylic acid, and the macropores are introduced via cryo-polymerization. By incorporating GM-CSF and tumor EVs into the macroporous hydrogel, a high number of DCs can be recruited in situ to process and present EV-encased antigens. These tumor antigen-presenting DCs can then traffic to lymphatic tissues to prime antigen-specific CD8+ T cells. This bioadhesive macroporous hydrogel system provides a new platform for in situ recruitment and modulation of DCs and the development of enhanced immunotherapies including tumor EV vaccines. We also envision the promise of this material system for drug delivery, tissue regeneration, long-term immunosuppression, and many other applications. The online version contains supplementary material available at 10.1007/s12195-023-00770-2.

Small-fibre neuropathy in leprosy the role of in vivo confocal microscopy: A cross-sectional study.

Background Leprosy (or Hansen's disease) continues to present considerable challenges regarding containment and early diagnosis. Leprosy is considered to be primarily a neural disease that first affects the sensory function of small fibres. Although the condition is well described in terms of clinical manifestations and histology, few studies have been undertaken to detect damage done to small-fibre sensory nerves. In vivo confocal microscopy is a useful tool for conducting a detailed evaluation of these structures, although its use in individuals affected by leprosy has still not been explored. Objective To evaluate in vivo confocal microscopy findings in Hansen's disease patients and their association with clinical variables relating to this disease. Method A cross-sectional case-series type study was carried out between October 2019 and May 2021, in Recife, Pernambuco, Brazil. Socio-demographic and clinical data were gathered from 21 patients with leprosy. The douleur neuropathique 4 neuropathic pain questionnaire was used to evaluate pain. In vivo confocal microscopy of the cornea was employed to evaluate the small-calibre fibres. Findings were compared with those for a control group of 23 healthy individuals. Results In relation to clinical parameters, 90.5% of the patients were classified as "multibacillary" according to the World Health Organization criteria, and 70% as dimorphic or borderline, in accordance with the Madrid classification. Around 52.4% had received a diagnosis after one year or less of living with the disease, while 95.2% presented alterations in small-fibre sensory function and 35% presented such alterations in the large fibre. Neuropathic pain was present in 81% of the patients. In vivo confocal microscopy found no statistically significant difference in mean age and distribution according to sex between the Hansen disease patients and the control group of healthy individuals. The median-of-means for dendritic cells and volume of sub-basal nerve fibres in the control group were used to test for normality. Both eyes of all leprosy patients examined contained higher number of dendritic cells than the median value and a volume of sub-basal nerve fibres lower than the mean. These differences were statistically significant (P < 0.001 and P < 0.001, respectively). Multibacillary individuals had a median number of dendritic cells two times that of paucibacillary individuals (P = 0.035). Limitations No association was found between the variables examined using in vivo confocal microscopy and clinical variables relating to small-fibre damage, the neuropathic pain questionnaire or alterations detected by the neurological examination. We believe, however, that Cochet-Bonnet esthesiometry of the cornea may have revealed such an association. Conclusion In vivo confocal microscopy is a useful diagnostic tool for detecting small fibre loss in individuals affected by leprosy and may constitute a useful addition to the range of tools available to help curb the effects of neuropathy in these patients.

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice.

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (>10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Non-transplantable cord blood units as a source for adoptive immunotherapy of leukaemia and a paradigm of circular economy in medicine.

Advances in immunotherapy with T cells armed with chimeric antigen receptors (CAR-Ts), opened up new horizons for the treatment of B-cell lymphoid malignancies. However, the lack of appropriate targetable antigens on the malignant myeloid cell deprives patients with refractory acute myeloid leukaemia of effective CAR-T therapies. Although non-engineered T cells targeting multiple leukaemia-associated antigens [i.e. leukaemia-specific T cells (Leuk-STs)] represent an alternative approach, the prerequisite challenge to obtain high numbers of dendritic cells (DCs) for large-scale Leuk-ST generation, limits their clinical implementation. We explored the feasibility of generating bivalent-Leuk-STs directed against Wilms tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME) from umbilical cord blood units (UCBUs) disqualified for allogeneic haematopoietic stem cell transplantation. By repurposing non-transplantable UCBUs and optimising culture conditions, we consistently produced at clinical scale, both cluster of differentiation (CD)34+ cell-derived myeloid DCs and subsequently polyclonal bivalent-Leuk-STs. Those bivalent-Leuk-STs contained CD8+ and CD4+ T cell subsets predominantly of effector memory phenotype and presented high specificity and cytotoxicity against both WT1 and PRAME. In the present study, we provide a paradigm of circular economy by repurposing unusable UCBUs and a platform for future banking of Leuk-STs, as a 'third-party', 'off-the-shelf' T-cell product for the treatment of acute leukaemias.

Abstract PO067: The immune cell infiltration and landscape predicts clinical outcomes in gynecologic cancers

Abstract Introduction: Immunotherapy has changed the standard of treatment for many cancers. However, only a small number of gynecologic cancer patients benefit from immunotherapy. The intratumoral immune landscapes are suggested as a predictor of the response to immunotherapies. However, there are no studies that provide a comprehensive immune characterization for gynecologic cancers. Aims: To characterize cellular compositions of the immune infiltrates and investigate if the immune landscape is a predictor for patient prognosis in gynecologic cancers. Methods: Clinical data for ovarian cancer, cervical cancer, and uterine cancer were downloaded via TCGA. RNA data are download from http://gdac.broadinstitute.org. Immune cell infiltration was analyzed using QuanTIseq and EPIC. Immune subtype clusters of patients were identified by Graph-based consensus clustering. Statistical analysis was conducted by GraphPad Prism. Results: Ovarian cancer had the highest percentage of total immune cells (approximately 21%, including CD4 T-Cells, Monocytes, CD8 T-Cells, Dendritic Cells, Macrophages, Neutrophils, NK Cells, B-Cells). Cervical cancer and uterine corpus endometrial carcinoma have lower percentages of immune cells with 17% and 16%, respectively. Furthermore, ovarian cancer had a significantly higher Monocyte and M2-liked Macrophage percentage, but lower percentage for CD8 T cells and Neutrophils compared to cervical cancer and uterine cancer. Cervical cancer had the highest percentage for M1-liked Macrophages and lowest CD4 T cells. Uterine cancer had the highest percentage of dendritic cells. In cervical cancer, higher cell infiltration of CD8 T-Cells and M2-liked Macrophages was associated with a better prognosis. In uterine cancer, patients with higher number of dendritic cells and CD8 T-Cells had significantly better clinical outcomes. However, higher CD4 T-cell infiltration was associated with poor prognosis in uterine cancer. Interestingly, patient’s survival was not affected by the infiltration of any individual immune cells which we analyzed in ovarian cancer. We identified and validated four immune subtypes associated with distinct immune cell infiltration in gynecologic cancers. Cervical and uterine cancer patients from an immune-desert subtype that had the least amount of lymphocyte infiltration and a high level of Monocyte had the worst prognosis. By contrast, Cervical and uterine cancer patients from an immune-warm subtype that had higher infiltration of CD8 T-cell, NK Cell, and dendritic cells had the best prognosis. However, the survival rate of ovarian cancer patients is similar among four different subtypes. Conclusion: Our study provides a conceptual framework to understand the tumor immune microenvironment of different gynecologic cancers. This work also suggests that the immune microenvironment should be considered for the design of combination treatment strategies and guiding the optimal selection of patients for immunotherapy. Citation Format: Wai Chung Chen, Tuo Hu, Chunbo He. The immune cell infiltration and landscape predicts clinical outcomes in gynecologic cancers [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO067.

FOXP3 in Melanoma with Regression: Between Tumoral Expression and Regulatory T Cell Upregulation.

Cutaneous melanoma is a significant immunogenic tumoral model, the most frequently described immune phenomenon being tumor regression, as a result of the interaction of tumoral antigens and stromal microenvironment. We present a retrospective cohort study including 52 cases of melanoma with regression. There were evaluated correlations of the most important prognostic factors (Breslow depth and mitotic index) with FOXP3 expression in tumor cells and with the presence of regulatory T cells and dendritic cells in the tumoral stroma. FOXP3 expression in tumor cells seems an independent factor of poor prognosis in melanoma, while regression areas are characterized by a high number of dendritic cells and a low number of regulatory T cells. FOXP3 is probably a useful therapeutical target in melanoma, since inhibition of FOXP3-positive tumor clones and of regulatory T cells could eliminate the ability of tumor cells to escape the immune defense of the host.

Automated dendritic cell generation in a closed system

Background & Aim Dendritic cells (DC) are an attractive vehicle for therapeutic manipulation and are used in antigen-pulsed autologous DC therapies, DC-stimulated autologous T cell therapies, and a variety of in vitro cellular immunology assays. Given the low abundance of DC in blood, they are typically generated ex vivo from monocytes or stem cell precursors in a manual culture process. The standard multiwell plate or flask culture used for DC generation requires several solution exchanges including cytokine refresh and wash steps. When DCs must be generated in a patient-specific manner, the multistep nature of the protocol presents significant logistical challenges for consistent results. To this end, the Corning MicroDEN® system relies upon medium perfusion to supply fresh cytokines throughout the duration of monocyte-derived DC generation. This study demonstrates performance of the MicroDEN system for immature DC (iDC) generation from a source of enriched monocytes. Methods, Results & Conclusion Monocytes were isolated from whole blood via Ficoll separation and CD14 magnetic bead purification. Monocyte differentiation to iDCs was achieved by a 6 day culture with CellGenix DC medium containing IL4 (350 U/mL;CellGenix), GM-CSF (350 U/mL;CellGenix), and 1% penicillin-streptomycin in both 6-well tissue culture-treated (TCT) multiwell plates (Corning) and the MicroDEN system. Flow cytometry data indicated that the iDCs from MicroDEN and 6-well plates were phenotypically comparable with regard to expression of CD209, CD80, CD83, CD86 and CD14. Allogeneic T cell proliferation assays and an antigen-specific assay demonstrated that iDCs generated via MicroDEN are functionally competent and can successfully generate a T cell response. A DOE-based approach was employed to characterize the relationship between perfusion flow rate, monocyte seeding density, and yield of dendritic cells. DCs generated by MicroDEN are functionally equivalent to DCs generated by the standard well plate culture. The larger surface area of MicroDEN cartridge allows generation of a higher number of dendritic cells in single run when compared to the standard 6-well plate, thereby reducing time and labor involved in harvesting cells from multiple plates. Higher perfusion flow rates enable the processing of larger numbers of seeded monocytes, upto a seeding density level of 1.3 million monocytes/cm2. MicroDEN is an effective tool to generate dendritic cells for applications ranging from cell-based assays to cell-based immunotherapies.

Diagnostic accuracy of pigmented labial macules by in vivo reflectance confocal microscopy and correlation among techniques

Pigmented labial macules (PLMs) are clinical, dermoscopic, and histopathologic challenges. To describe and evaluate the utility of reflectance confocal microscopy (RCM) in PLMs and to establish a correlation between dermoscopy, RCM, histopathology, and immunohistochemistry. Prospective study of PLMs from 4 tertiary referral dermatology centers. The study included 51 biopsy specimen-proven PLMs. Dermoscopic, RCM images, and histopathologic preparations were evaluated for malignant criteria. Diagnostic accuracy of RCM for melanoma diagnosis, RCM Lip Score previously reported, and κ values between techniques were calculated. Included were 5 melanomas and 46 benign PLMs. Dermoscopically, melanomas exhibited more frequently ≥3 colors and ≥3 structures. With RCM, pagetoid spreading, epithelial disarray, continuous proliferation of atypical cells around papillae, nonhomogeneously distributed papillae, marked cellular atypia, and a higher number of dendritic cells per papillae were more frequent in melanomas. The RCM Lip Score was significantly higher in malignant lesions. Good κ values were observed in most of the evaluated features. A perfect sensitivity and specificity was obtained combining dermoscopy and RCM. A low number of melanomas were obtained. RCM improves lip melanoma diagnosis, and the RCM Lip Score represents a useful tool for the evaluation of a PLM.

In Vivo Reflectance Confocal Microscopy for the Diagnosis of Melanoma and Melanotic Macules of the Lip

ImportanceBenign melanotic macules (MAC) are the most frequent cause of lip pigmentation and sometimes difficult to differentiate from lip melanoma (MEL).ObjectivesTo report in vivo reflectance confocal microscopy (RCM) features of normal lips of different phototypes and to identify features that assist in distinguishing MEL from MAC using dermoscopy and RCM.Design, Setting, and ParticipantsFor this retrospective observational study, 2 groups of patients from 2 tertiary referral centers for melanoma (Sydney Melanoma Diagnostic Centre and Melanoma Institute Australia) were recruited between June 2007 and January 2015. Group 1 included patients with normal lips and different phototypes, and Group 2 consisted of patients with MAC and MEL; RCM and dermoscopy were used for lips analysis.Main Outcomes and MeasuresOverall, 92 RCM features were correlated with clinical history, dermoscopic images, and histopathology in all patients with MEL and 5 patients with MAC.ResultsImages from the vermillion and/or mucosal part of the lip were recorded from 10 patients with clinically normal lips (mean [SD] age, 34.5 [6.1] years), 16 patients with MAC (mean [SD] age, 49.6 [17.9] years), and 5 patients with 6 cases of MEL (1 patient had a recurrent lesion; mean [SD] age, 56.2 [15.5] years). In normal lips, the draped pattern—a previously described MAC RCM feature—was identified in all cases. In MEL, the following findings were frequent and significantly different from MAC: epidermal disarray; pagetoid infiltration of dendritic and/or round cells; a nonspecific architectural pattern at the dermoepidermal junction (DEJ); nonhomogenously distributed papillae; continuous (lentiginous) proliferation of cells with marked atypia at the DEJ, especially in interpapillary spaces; a higher number of dendritic cells (especially roundish); and atypical round cells at the DEJ. The cellular body area of dendritic cells was about the double in MEL compared with MAC. An RCM lip algorithm was developed that provided 100% sensitivity and 88% specificity for the diagnosis of MEL of the vermillion and mucosal part of the lip. With dermoscopy, MAC were correctly classified as benign in 13 of 16 cases (81%) and MEL were classified as equivocal or malignant in 5 of 6 cases (83%).Conclusions and RelevanceReflectance confocal microscopy can assist in the differential diagnosis of lip MEL and MAC. An RCM Lip Score that we developed based on study results is proposed and needs to be validated on an independent data set.

Correction: Depletion of Regulatory T Cells Induces High Numbers of Dendritic Cells and Unmasks a Subset of Anti-Tumour CD8+CD11c+ PD-1lo Effector T Cells.

[This corrects the article DOI: 10.1371/journal.pone.0157822.].

Depletion of Regulatory T Cells Induces High Numbers of Dendritic Cells and Unmasks a Subset of Anti-Tumour CD8+CD11c+ PD-1lo Effector T Cells.

Natural regulatory T (Treg) cells interfere with multiple functions, which are crucial for the development of strong anti-tumour responses. In a model of 4T1 mammary carcinoma, depletion of CD25+Tregs results in tumour regression in Balb/c mice, but the mechanisms underlying this process are not fully understood. Here, we show that partial Treg depletion leads to the generation of a particular effector CD8 T cell subset expressing CD11c and low level of PD-1 in tumour draining lymph nodes. These cells have the capacity to migrate into the tumour, to kill DCs, and to locally regulate the anti-tumour response. These events are concordant with a substantial increase in CD11b+ resident dendritic cells (DCs) subsets in draining lymph nodes followed by CD8+ DCs. These results indicate that Treg depletion leads to tumour regression by unmasking an increase of DC subsets as a part of a program that optimizes the microenvironment by orchestrating the activation, amplification, and migration of high numbers of fully differentiated CD8+CD11c+PD1lo effector T cells to the tumour sites. They also indicate that a critical pattern of DC subsets correlates with the evolution of the anti-tumour response and provide a template for Treg depletion and DC-based therapy.

Abstract A044: Initial phase I/IIa trial results of an autologous tumor lysate + yeast cell wall particles + dendritic cells vaccine (TLPLDC) in patients with solid tumors

Abstract Introduction: The majority of tumor vaccines (vax) target tumor-specific/associated antigens with a variety of delivery systems/immunoadjuvants. As opposed to testing an “off the shelf” vax to treat a subset of patients (pts), our approach is tailored to the specific pt but applicable to all pts, all tumors. Our vax uses autologous dendritic cells (DC) loaded with the full range of tumor antigens from a pt's cancer. Our particle loading system is highly efficient, reducing the need for large tumor volume, high numbers of DC or long culture times. The autologous tumor lysate (TL) is loaded into yeast cell wall particles (YCWP) that are naturally and efficiently taken up into isolated autologous DC. These autologous TL, particle-loaded, DC (TLPLDC) can be produced in 48hrs. Here, we describe the results of the TLPLDC vax in the first 20 pts treated. Methods: We are conducting an ongoing open label phase I/IIa trial enrolling pts with any solid tumor and treating them with autologous TLPLDC specifically created in our phase I trial center under regulatory oversight. Enrollment criteria include pts with all solid tumors of all stages, with ECOG status ≤1, and &amp;gt;4 months life expectancy. Exclusion criteria are chronic immunosuppression or viral infection and pregnancy. DC are isolated from the pt's peripheral blood monocytes from a 120ml blood draw; cytokines are added to generate immature monocyte-derived DC. A minimum of 1 mg frozen tumor is required from surgery or biopsy. Tumor cells are lysed by multiple freeze/thaw cycles in buffer to produce TL. YCWP are prepared by NaOH digestion of all non-cell wall material from Saccharomyces cerevisiae, followed by washings. These hollow YCWP are loaded with concentrated TL solution via absorption, then freeze dried. Tetanus helper peptide and CpG oligonucleotides are similarly loaded into the YCWP as immunoadjuvants. TLPLDC are generated by incubating TL-loaded YCWP in the presence of DC and cytokines. After phagocytosis, final TLPLDC are frozen in single dose (1x106 cells/dose) vials. After QA/QC testing and lot-release, pts receive 4 monthly intradermal inoculations and then boosters every 6 months for as long as doses last. Trial endpoints are safety and efficacy using CTCAE and RECIST criteria, respectively. Results: To date, 20 pts have been enrolled to the TLPLDC trial with a median follow-up of 7 mos. The study population is 60% male, 90% Caucasian, 95% stage III/ IV disease (dz), with a median age of 58 years. Tumor types include chondrosarcoma, melanoma, astrocytoma, glioblastoma, Ewing's sarcoma, and renal cell, ovarian, breast, esophageal, pancreatic, squamous cell, bladder, and small cell lung carcinomas. Of the 20 pts, 17 had measurable dz on initiation of treatment, and 3 were vaccinated adjuvantly. Twelve patients completed treatment, 6 are in treatment, and 2 are consented but not initiated. The 12 pts who have completed treatment received a median of 4 inoculations, and no grade ≥3 toxicities have been noted. Flu-like symptoms have been seen in 20%. Of the 17 pts with measurable dz, 10 have completed treatment with 6 demonstrating clinical benefit (1 CR, 2 PR, 3 SD). In the 3 adjuvant pts, 2 have completed treatment, and all 3 remain dz-free at a median follow- up of 10 months. Conclusions: Current results from this ongoing phase I/ IIa trial indicate that the autologous TLPLDC vax is well-tolerated and safe. The vax appears to have clinical activity in a variety of solid tumors to include melanoma with a complete response. Based on our prior studies with a similar vax and these initial results, TLPLDC may be most efficacious in the minimal residual dz setting; therefore, an adjuvant randomized phase IIb trial in stage III/IV (resected) melanoma pts has been initiated. Citation Format: Julia M. Greene, Diane F. Hale, Erika J. Schneble, Xianzhong Yu, Pauline Nichol, Sook Yin, Thomas Wagner, George E. Peoples. Initial phase I/IIa trial results of an autologous tumor lysate + yeast cell wall particles + dendritic cells vaccine (TLPLDC) in patients with solid tumors. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A044.

Laser-assisted intradermal delivery of adjuvant-free vaccines targeting XCR1+ dendritic cells induces potent antitumoral responses.

The development of vaccines inducing efficient CD8(+) T cell responses is the focus of intense research. Dendritic cells (DCs) expressing the XCR1 chemokine receptor, also known as CD103(+) or CD8α(+) DCs, excel in the presentation of extracellular Ags to CD8(+) T cells. Because of its high numbers of DCs, including XCR1(+) DCs, the skin dermis is an attractive site for vaccine administration. By creating laser-generated micropores through the epidermis, we targeted a model protein Ag fused to XCL1, the ligand of XCR1, to dermal XCR1(+) DCs and induced Ag-specific CD8(+) and CD4(+) T cell responses. Efficient immunization required the emigration of XCR1(+) dermal DCs to draining lymph nodes and occurred irrespective of TLR signaling. Moreover, a single intradermal immunization protected mice against melanoma tumor growth in prophylactic and therapeutic settings, in the absence of exogenous adjuvant. The mild inflammatory milieu created in the dermis by skin laser microporation itself most likely favored the development of potent T cell responses in the absence of exogenous adjuvants. The existence of functionally equivalent XCR1(+) dermal DCs in humans should permit the translation of laser-assisted intradermal delivery of a tumor-specific vaccine targeting XCR1(+) DCs to human cancer immunotherapy. Moreover, considering that the use of adjuvants in vaccines is often associated with safety issues, the possibility of inducing protective responses against melanoma tumor growth independently of the administration of exogenous adjuvants should facilitate the development of safer vaccines.

Dendritic cells and coronary collateral circulation in coronary heart disease

To determine the relationship between the number,phenotype and functional status of dendritic cells (DCs) and coronary collateral circulation (CCC) in coronary heart disease (CHD). Forty patients with severe coronary stenosis were recruited and divided into a CCC formation group (Group A, n=22) and a non-CCC formation group (Group B, n=18). Density gradient centrifugation was applied to separate the mononuclear cells (MNCs) from coronary artery blood samples, and MNCs were cultured and proliferated in vitro. The morphology of DCs was observed under converted microscope. The number of harvested cells and DCs was counted by hematocytometer. Flow cytometry was applied to investigate the phenotype and the mean fluorescence intensity (MFI). Mixed lymphocyte reaction was used to test the function of DCs to stimulate the proliferation of T lymphocytes. Stimulation index (SI) was calculated and compared. (1) After in vitro proliferation, DCs were cultured successfully from the mononuclear cells from coronary artery blood samples and the morphology of DCs was not different in the 2 groups. (2) The number of mononuclear cells (MNC no) was (3.95+/-1.41)*10(6), in the CCC group and (2.76+/-0.92)*10(6) in the non-CCC group. The MNC number was significantly increased in the CCC group (P=0.003). (3) The number of DCs was (1.54+/-0.96)*10(6) in the CCC group, and (0.99+/-0.46)*10(6) in the non-CCC group (P=0.033). (4)There was no statistical significance in the percent of CD1a+, CD1a+CD80+, CD1a+CD83+, CD1a+CD86+ cells, and MFI in the 2 groups (P>0.05). (5) SI was 4.96+/-2.30 in the CCC group, whereas 2.66+/-1.04 in the non-CCC group. The SI in the CCC group increased significantly(P=0.0003). In CHD patients with severe coronary stenosis, patients with CCC formation have higher number of DCs and stronger potential of T lymphocyte stimulation.

Assessment of Migration of HIV-1-Loaded Dendritic Cells Labeled with 111 In-Oxine used as a Therapeutic Vaccine in HIV-1-Infected Patients

Monocyte-derived dendritic cells (DCs) loaded with heat-inactivated HIV are used in therapeutic immunizations. It is not known whether they migrate in vivo to lymph nodes. We used an (111)In-oxine-labeled DC (ILDC) method to visualize the migration of DCs. The activity, time and incubation medium were investigated to obtain the highest cellular viability and radiolabeling yield. A trypan-blue exclusion test was used to determine the cellular viability. In five patients, 2 x 10(6) ILDCs were injected subcutaneously in the arm. An initial dynamic study was performed during the first 5 min after injection. This was followed by static acquisitions at several time points, using a high-resolution (general electric) gamma-camera and quantifying the activity at regions of interest drawn on the injection point. The sensitivity of the gamma-camera was evaluated. The highest number of viable DCs (>83%) and the best radiolabeling yield (>70%) were obtained with 1.11 MBq (111)In-oxine, after 10 min of incubation at 37 degrees C in sodium chloride solution 0.9%. We did not observe migration of ILDCs to local lymph nodes in any patient. However, focal uptake at the place of injection continued during the study period. We observed a higher than expected loss of activity from the injection point (median A(t)/A(0) = 0.60 at day 2), which correlated with an increase in total cytotoxic T lymphocytes (CD8(+) and granzyme B(+) cells) in the lypmphoid tissue observed after immunization (R(2) = 0.92, p = 0.03). If more than 20,000 ILDCs had migrated, they could have been detected. In future trials, a higher number of DCs or alternative methods should be used to assess the migration of DCs to lymph nodes.

Ex vivo expanded telomerase-specific T cells are effective in an orthotopic mouse model for pancreatic adenocarcinoma.

Telomerase activity is over-expressed in nearly all pancreatic carcinomas, but not in chronic pancreatitis. Here, we investigated various protocols for expansion of telomerase-specific T cells for adoptive cell transfer and their use in a syngeneic pancreatic carcinoma mouse model. Telomerase-specific T cells were generated by stimulation of splenocytes from peptide-immunized donor mice with either interleukin (IL)-2, IL-15, artificial antigen-presenting cells, anti-signalling lymphocyte activation molecule (SLAM) microbeads or allogeneic dendritic cells in combination with a limited dilution assay. T cells were tested for antigen specificity in vitro and for anti-tumour activity in syngeneic mice with orthotopically implanted tumours pretreated with cyclophosphamide. The immune cells from recipients were immunophenotyped. During a period of 2 weeks, the expansion approach using IL-2 was very successful in generating a high number of telomerase-specific CD8(+) T cells without losing their function after adoptive cell transfer. Significantly slower tumour growth rate and less metastasis were observed after adoptively transferring telomerase specific CD8(+) T cells, expanded using IL-2. Further investigations showed that anti-tumour efficacy was associated with a significant shift from naive CD8(+) T cells to CD8(+) central memory T cells, as well as recruitment of a high number of dendritic cells. Remarkable amounts of telomerase-specific T cells were detectable in the tumour. Generation of telomerase-specific T cells is feasible, whereat IL-2-based protocols seemed to be most effective and efficient. Antigen-specific T cells showed significant cytotoxic activity in a syngeneic, orthotopic mouse model, whereas central memory T cells but not effector memory T cells appear to be of high importance.

Intracerebral granulocyte‐macrophage colony‐stimulating factor induces functionally competent dendritic cells in the mouse brain

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor and a proinflammatory cytokine. While GM-CSF is lacking in normal brain tissue, it is expressed under pathological conditions and correlates with the presence of dendritic cells (DC). However, the role of GM-CSF for the onset of immune responses in the brain is still unclear. To analyze the role of GM-CSF for the induction and functional activity of immune cells in the brain, we performed chronic intracerebroventricular administration of GM-CSF to the brains of adult mice. After GM-CSF administration, intracerebral leukocytes (ICL) were characterized by means of flow cytometry, immunohistochemistry, and an ex vivo functional assay. GM-CSF treatment significantly increased the number of leukocytes expressing high levels of CD45, indicative of peripheral, blood-derived cells. The infiltrating cells were preferentially DC of the myeloid lineage (CD45(high) CD11c+ CD11b+) with an activated phenotype characterized by upregulated expression of MHCII and the costimulatory ligand CD80. Furthermore, DC from GM-CSF treated mice were fully competent to activate naive allogeneic T cells in a mixed leukocyte reaction. In contrast, intracerebroventricular IFN-gamma administration stimulated MHCII expression on cells resembling resident microglia, but did not induce comparable presence of DC. Taken together, intracerebroventricular GM-CSF treatment results in high numbers of DC in the brain. Moreover, these GM-CSF-induced DC display an activated phenotype and exhibit the capacity to act as fully competent DC even without a further inflammatory stimulus.

CD83 +CCR7 − Dendritic Cells Accumulate in the Subepithelial Dome and Internalize Translocated Escherichia coli HB101 in the Peyer's Patches of Ileal Crohn's Disease

Recurrent Crohn's disease originates with small erosions in the follicle-associated epithelium overlying the Peyer's patches. Animal studies have illustrated mucosal immune regulation by dendritic cells located in the subepithelial dome. The aim of this study was to characterize the dendritic cells at this specific site in patients with Crohn's disease. Ileal tissues were obtained after surgery performed on Crohn's patients; ileal samples from noninflammatory bowel disease and ulcerative colitis served as standard and inflammatory controls, respectively. Flow cytometry of isolated intestinal mononuclear cells showed a larger subset of dendritic cells in Crohn's samples compared with controls. This finding was corroborated by confocal microscopy, showing enhanced infiltrates of cells positive for the dendritic cell markers, DC-SIGN(+) and CD83(+), in the subepithelial dome. Moreover, the CD83(+) cells in Crohn's tissues showed reduced expression of the lymph node migratory receptor, CCR7, possibly contributing to the high numbers of dendritic cells. After exposure to nonpathogenic Escherichia coli in Ussing chambers, dendritic cells in the subepithelial dome of Crohn's disease demonstrated increased co-localization with translocated bacteria. Immunohistochemical results revealed that DC-SIGN(+) cells in Crohn's tissues were found to express toll-like receptor 4 and produce tumor necrosis factor-alpha. In conclusion, nonmigrating dendritic cells that accumulate in the subepithelial dome and internalize nonpathogenic bacteria may be important for the onset and perpetuation of mucosal inflammation in Crohn's disease.

Immunological markers after long-term treatment interruption in chronically HIV-1 infected patients with CD4 cell count above 400 × 106 cells/l

To analyse immunological markers associated with CD4+ lymphocyte T-cell count (CD4+) evolution during 12-month follow-up after treatment discontinuation. Prospective observational study of chronically HIV-1 infected patients with CD4+ above 400 x 10(6) cells/l. CD4+ changes took place in two phases: an initial rapid decrease in the first month (-142 x 10(6) cells/l on average), followed by a slow decline (-17 x 10(6) cells/l on average) The second slope of CD4+ decline was not correlated with the first and only baseline plasma HIV RNA was associated with it. The decline in CD4+ during the first month was steeper in patients with higher CD4+ and weaker plasma HIV RNA baseline levels. Moreover, the decline was less pronounced (P < 10(-4)) in patients with CD4+ nadir above 350 x 10(6) cells/l (-65 x 10(6) cells/l per month) in comparison with those below 350 x 10(6) cells/l (-200 x 10(6) cells/l per month). A high number of dendritic cells (DCs) whatever the type was associated with high CD4+ at the time of treatment interruption and its steeper decline over the first month. Moreover, the myeloid DC level was stable whereas the lymphoid DC count, which tended to decrease in association with decrease in CD4+, was negatively correlated with the HIV RNA load slope. The results support the use of the CD4+ nadir to predict the CD4+ dynamic after treatment interruption and consideration of the CD4+ count after 1-month of interruption merely reflects the 12-month level of CD4+. Although DCs seem to be associated with the CD4+ dynamic, the benefit of monitoring them has still to be defined.

Lack of dendritic cell mobilization into the peripheral blood of cancer patients following standard- or high-dose chemotherapy plus granulocyte-colony stimulating factor.

Dendritic cells (DC), the most specialized antigen-presenting cells, can be detected in the peripheral blood (PB) and divided into two subsets of populations, DC1 and DC2, endowed with different functions. The aim of this study was to evaluate the effect on DC release and on their subsets of three regimens utilized to mobilize CD34+ cells into the PB in cancer patients and in normal CD34+ cell donors. The mobilizing sequences were: standard-dose epirubicin+taxol+granulocyte-colony-stimulating factor (G-CSF; 15 patients with advanced breast cancer), high-dose cyclophosphamide (CTX)+G-CSF (10 patients with breast cancer patients and 7 with non-Hodgkin's lymphoma, NHL), and G-CSF alone (5 normal donors of CD34+ cells for allogeneic transplantation). Comparative data were obtained from the steady-state PB of 20 healthy volunteers. For flow cytometric analysis, DC were gated as negative for specific lineage markers (CD3, CD11b, CD14, CD16, CD56, CD19, CD20, CD34) and positive for HLA-DR. The DC1 and DC2 subsets were defined as CD11c and CDw123 positive, respectively. The percentages of DC at baseline and the time of CD34+ cell peak were: 0.48 and 0.51 for standard-dose chemotherapy (CT); 0.55 and 0.63 for breast cancer after high-dose CTX+G-CSF; 0.53 and 0.71 for NHL after high-dose CTX+G-CSF; and 0.51 and 0.54 for normal donors of CD34+ cells after G-CSF alone (all p=n.s.). Mean DC1/DC2 ratios in each study group at the time of CD34+ cell peak were 0.10, 0.12, and 0.18, respectively. Finally, in the group of healthy volunteers, the percentage of circulating DC was 0.95 and the mean DC1/DC2 ratio was 1.28. To our knowledge, this is the first report that demonstrates that both standard-dose or high-dose CT, when utilized together with G-CSF, do not induce DC mobilization into the PB, whereas a reversed DC1/DC2 ratio is observed. Furthermore, a lack of significant DC mobilization after G-CSF alone was also seen, in contrast to what was previously observed by others. These data should be taken in account when evaluating clinical correlations between DC number and CPC engraftment in both the transplantation setting, when monitoring the effects on the immune system of combinations of new drugs and/or cytokines, and when high numbers of DC are required for both experimental and clinical applications.