Generation of Leukemia-Derived Dendritic Cells (DCleu) as a Basis for Specific Immunotherapy in Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2547-2547
Author(s):  
Helga Schmetzer ◽  
Christoph Schmid ◽  
Susanne Kufner ◽  
Renate Pelka-Fleischer ◽  
Tanja Kroell ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cell Response ◽  
T Cell Response ◽  
Aberrant Expression ◽  
Immune Phenotype ◽  
Cytogenetic Aberrations ◽  
Stimulation Of

Abstract The presentation of leukemic antigens can be improved in AML and MDS by in vitro generation of dendritic cells of leukemic origin (DCleu), thereby creating a platform for the generation of leukemia-specific cytotoxic lymphocytes (CTL). To further investigate this approach, we developed a serum-free culture system using MCM-Mimic medium (X-vivo + GM-GSF + IL-4 + FL + IL1β + IL-6 + TNFα + PGE2) for differentiation of malignant myeloid blasts to DCleu. Periperal blood mononuclear cells (PB-MNC) were obtained from 100 AML and 55 MDS-patients. Samples contained a mean of 59%/and 6% of blasts, respectively. After 14 days, cultures contained on average 34% (AML) and 20%(MDS) DC. DC yields were best in monocytic subtypes (AML M4, M5 and CMML). Cytogenetic aberrations of the leukemia had no influence. The leukemic origin of cultured DC was demonstrated using a acombined FISH/immune phenotyping assay (FISH/IPA): In cells showing a characteristic DC morphology and immune phenotype, FISH was used to detect specific cytogenetic aberrations identified in the leukemic population at time of diagnosis. Alternativly, DCleu were identified by detecting coexpression of DC markers and a leukemia-specific immune phenotype (as defined by aberrant expression of lineage markers or a characteristic CD34+/CD117+ phenotype in MDS). However, in most cases not all leukemic blasts in a given sample could be differentiated, since on average 47% of clonal cells did not acquire a DC-like immunophenotype. Vice versa, not all DC identified at the end of the culture period were DCleu. The capacity of DCleu to elicit a specific T-cell response was demonstrated by upregulation of contact-molecules, responsible for DC/T-cell contact, by DC-activated T-cell proliferation and by the capacity of DC-activated T-cells to specifically lyse naive blasts. In 6%/31% of AML/MDS-cases, <10% DC could be generated. Therefore, other DC-culture-assays were compared with respect to DC harvest, to identify the best method for DC-generation in each individual patient. Compared to `MCM-Mimic`, harvest of DC could be improved by `CA-Ionophore-media`(A23187 + Il4) in 4 of 7 cases, whereas in 3 cases, MCM led to higher DC-yields. In conclusion, DCleu can be generated in the majority of patients with AML and MDS. To optimize DC harvest for in vitro and in vivo use, different culture assays should be compared in each individual case. DCleu are able to elicit a specific T-cell response in vitro. Nevertheless, cultures containing DCleu also contain relevant numbers of undifferentiated blasts and DC of non-leukemic origin. These cells may represent an obstacle for the clinical use of DCleu, since they may cause specific anergy or unspecific stimulation of effector T-cells. Improvement of culture conditions for generation of DCleu, and methods to separate DCleu before stimulation of effector cells will be required, before clinical trials are feasible.

Transfection of Dendritic Cells with In Vitro Transcribed RNA Induces Polyclonal CMV-Specific CD8+ and CD4+ Mediated T Cell Responses.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1354-1354
Author(s):  
Annkristin Heine ◽  
Tobias Holderried ◽  
Frank Grünebach ◽  
Silke Appel ◽  
Markus M. Weck ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cell Response ◽  
Cytotoxic T Cells ◽  
T Cell Responses ◽  
T Cell Response ◽  
Cell Responses ◽  
Rna Transfection

Abstract Transfection of dendritic cells (DC) with in vitro transcribed RNA was shown to be a highly efficient method to generate antigen specific T cells, probably due to the induction of a polyclonal T cell response directed against multiple antigens presented on different HLA allels. However, the experimental evidence of this assumption remains to be demonstrated. To accomplish this, we used monocyte derived DC that were electroporated with RNA coding for the CMV pp65 antigen. The induction and expansion of antigen specific CD8+ and CD4+ T cells was assessed using a pannel of peptides derived from this antigen and presented on HLA-A2, -A1, -A11, -A24, -B35 and -B7 in IFN-g ELISPOT, 51Cr-release and proliferation assays. Autologous DC generated from CMV positive healthy donors were pulsed with peptides or transfected with pp65 RNA and utilized as stimulators. Autologous purified CD8+ and CD4+ lymphocytes were used as effector cells. By applying this approach we found that transfection of DC with pp65 RNA induces an expansion of polyclonal CD8+ mediated T cell responses that recognized peptide antigens presented on different HLA molecules. These in vitro generated cytotoxic T cells were able to efficiently lyse DC pulsed with pp65 derived peptides or transfected with the cognate RNA in an antigen specific manner after several in vitro restimulations. Furthermore, this experimental approach allowed the identification of the immunodominace of T cell epitopes presented upon RNA transfection. The HLA-2 directed responses were more pronounced as compared to the HLA-A1, -A11, -A24 or -B35 allels. In contrast, in 7 out of 7 HLA-A2 and HLA-B7 positive donors B7-peptides elicited a stronger T cell response than the A2-peptide, indicating the immunodominance of HLA-B7 epitopes. Interestingly, transfection of DC with pp65 RNA resulted in the induction of CD4+ antigen specific T cells that produced IFN-g and proliferated upon stimulation with transfected DC. In the next set of experiments we analyzed the possible induction of CMV specific T cells that recognize epitopes deduced from different antigens. Co-transfection of DC with a mixture of RNAs coding for the CMV pp65 and IE1 antigens elicited polyclonal T lymphocytes specific for peptides derived from both antigens. More importantly, polyclonal cytotoxic T cells could be elicited in peripheral blood of 2 out of 3 CMV negative donors demonstrating the efficiency of this approach. Our results demonstrate that DC transfected with RNA can elicit polyclonal T cell responses and have implications for the development of immunotherapeutic strategies to target viral or tumor associated antigens.

scholarly journals Human Cytomegalovirus (HCMV)-Specific CD4+ T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro

2017 ◽  
Vol 91 (6) ◽  
Author(s):  
Sarah E. Jackson ◽  
George X. Sedikides ◽  
Gavin M. Mason ◽  
Georgina Okecha ◽  
Mark R. Wills
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Human Cytomegalovirus ◽  
Immune Cells ◽  
Cell Response ◽  
T Cell Response ◽  
Cell Responses ◽  
Infected Cells

ABSTRACT Human cytomegalovirus (HCMV) infection and periodic reactivation are generally well controlled by the HCMV-specific T cell response in healthy people. While the CD8+ T cell response to HCMV has been extensively studied, the HCMV-specific CD4+ T cell effector response is not as well understood, especially in the context of direct interactions with HCMV-infected cells. We screened the gamma interferon (IFN-γ) and interleukin-10 (IL-10) responses to 6 HCMV peptide pools (pp65, pp71, IE1, IE2, gB, and US3, selected because they were the peptides most frequently responded to in our previous studies) in 84 donors aged 23 to 74 years. The HCMV-specific CD4+ T cell response to pp65, IE1, IE2, and gB was predominantly Th1 biased, with neither the loss nor the accumulation of these responses occurring with increasing age. A larger proportion of donors produced an IL-10 response to pp71 and US3, but the IFN-γ response was still dominant. CD4+ T cells specific to the HCMV proteins studied were predominantly effector memory cells and produced both cytotoxic (CD107a expression) and cytokine (macrophage inflammatory protein 1β secretion) effector responses. Importantly, when we measured the CD4+ T cell response to cytomegalovirus (CMV)-infected dendritic cells in vitro, we observed that the CD4+ T cells produced a range of cytotoxic and secretory effector functions, despite the presence of CMV-encoded immune evasion molecules. CD4+ T cell responses to HCMV-infected dendritic cells were sufficient to control the dissemination of virus in an in vitro assay. Together, the results show that HCMV-specific CD4+ T cell responses, even those from elderly individuals, are highly functional and are directly antiviral. IMPORTANCE Human cytomegalovirus (HCMV) infection is carried for a lifetime and in healthy people is kept under control by the immune system. HCMV has evolved many mechanisms to evade the immune response, possibly explaining why the virus is never eliminated during the host's lifetime. The dysfunction of immune cells associated with the long-term carriage of HCMV has been linked with poor responses to new pathogens and vaccines when people are older. In this study, we investigated the response of a subset of immune cells (CD4+ T cells) to HCMV proteins in healthy donors of all ages, and we demonstrate that the functionality of CD4+ T cells is maintained. We also show that CD4+ T cells produce effector functions in response to HCMV-infected cells and can prevent virus spread. Our work demonstrates that these HCMV-specific immune cells retain many important functions and help to prevent deleterious HCMV disease in healthy older people.

scholarly journals Immunization with Apoptotic Phagocytes Containing Histoplasma capsulatum Activates Functional CD8+T Cells To Protect against Histoplasmosis

2011 ◽  
Vol 79 (11) ◽  
pp. 4493-4502 ◽  
Author(s):  
Shih-Hung Hsieh ◽  
Jr-Shiuan Lin ◽  
Juin-Hua Huang ◽  
Shang-Yang Wu ◽  
Ching-Liang Chu ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Response ◽  
Cell Activation ◽  
Histoplasma Capsulatum ◽  
T Cell Response ◽  
Content Type

ABSTRACTWe have previously revealed the protective role of CD8+T cells in host defense againstHistoplasma capsulatumin animals with CD4+T cell deficiency and demonstrated that sensitized CD8+T cells are restimulatedin vitroby dendritic cells that have ingested apoptotic macrophage-associatedHistoplasmaantigen. Here we show that immunization with apoptotic phagocytes containing heat-killedHistoplasmaefficiently activated functional CD8+T cells whose contribution was equal to that of CD4+T cells in protection againstHistoplasmachallenge. Inhibition of macrophage apoptosis due to inducible nitric oxide synthase (iNOS) deficiency or by caspase inhibitor treatment dampened the CD8+T cell but not the CD4+T cell response to pulmonaryHistoplasmainfection. In mice subcutaneously immunized with viableHistoplasmayeasts whose CD8+T cells are protective againstHistoplasmachallenge, there was heavy granulocyte and macrophage infiltration and the infiltrating cells became apoptotic. In mice subcutaneously immunized with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled apoptotic macrophages containing heat-killedHistoplasma, the CFSE-labeled macrophage material was found to localize within dendritic cells in the draining lymph node. Moreover, depleting dendritic cells in immunized CD11c-DTR mice significantly reduced CD8+T cell activation. Taken together, our results revealed that phagocyte apoptosis in theHistoplasma-infected host is associated with CD8+T cell activation and that immunization with apoptotic phagocytes containing heat-killedHistoplasmaefficiently evokes a protective CD8+T cell response. These results suggest that employing apoptotic phagocytes as antigen donor cells is a viable approach for the development of efficacious vaccines to elicit strong CD8+T cell as well as CD4+T cell responses toHistoplasmainfection.

scholarly journals Dendritic cells transduced by multiply deleted HIV-1 vectors exhibit normal phenotypes and functions and elicit an HIV-specific cytotoxic T-lymphocyte response in vitro

Blood ◽  
2000 ◽  
Vol 96 (4) ◽  
pp. 1327-1333 ◽  
Author(s):  
Andreas Gruber ◽  
June Kan-Mitchell ◽  
Kelli L. Kuhen ◽  
Tetsu Mukai ◽  
Flossie Wong-Staal
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Ex Vivo ◽  
T Cell Response ◽  
Adoptive T Cell Therapy ◽  
Cytotoxic T Cell ◽  
T Cell Therapy ◽  
Hiv 1

Abstract Dendritic cells (DCs) genetically modified to continually express and present antigens may be potent physiologic adjuvants for induction of prophylactic or therapeutic immunity. We have previously shown that an env and nef deleted HIV-1 vector (HIV-1ΔEN) pseudotyped with VSV-G transduced monocyte-derived macrophages as well as CD34+ precursors of DCs. Here we extended these findings with HIV-1ΔEN to highly differentiated human DCs derived in culture from circulating monocytes (DCs). In addition, a new vector derived from HIV-1ΔEN but further deleted in its remaining accessory genes vif, vpr, and vpu(HIV-1ΔEN V3) was also tested. Both vectors efficiently transduced DCs. Transduction of DCs did not significantly alter their viability or their immunophenotype when compared with untransduced DCs. Furthermore, the phagocytic potential of immature DCs, as well as their ability to differentiate into mature DCs capable of stimulating T-cell proliferation, was not affected. Finally, DCs transduced by the HIV-1ΔEN vector were able to elicit a primary antiviral cytotoxic T-cell response in autologous CD8 T cells. These results suggest that HIV-1–based vectors expressing viral antigens may be useful for in vivo active immunization as well as ex vivo priming of cytotoxic T cells for adoptive T-cell therapy.

Cultured Human B Cell APCs Expanded Using Il-4 and CD40 Ligand Preferentially Promote a Type 2 T Cell Response to Alloimmune Stimulation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2668-2668
Author(s):  
Abdul Tawab ◽  
Yoshiyuki Takahashi ◽  
Childs Richard ◽  
Kurlander J. Roger
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Response ◽  
Cd40 Ligand ◽  
T Cell Response ◽  
Ifn Γ

Abstract In vitro stimulation of human peripheral blood B cells with recombinant IL-4 and CD40 ligand (CD40L) markedly increases their expression of MHC and costimulatory molecules, thus enhancing antigenic peptide presentation to T cells. Because these cells proliferate extensively in vitro (unlike monocytes or dendritic cells), they represent a promising and convenient reagent for the generation and maintenance of antigen-specific T cells for use in a variety of experimental or therapeutic settings. However, the impact of this type of B cell APC on cytokine production by responder T cells has hitherto not been examined. To address this issue, we stimulated normal human T cells with either allogeneic B cells (generated in vitro) or with MNCs obtained from the same donor. After 7 days, T cells were washed and re-challenged with the same APCs. The resulting alloreactive cytokine response was measured using quantitative ELISPOT methods and expressed as the frequencies of IFN-γ, IL-4, and IL-5 producing cells per thousand responder cells added. B cell- and MNC-primed cell lines both produced vigorous lymphokine responses, but B cell-stimulated T cells consistently produced more IL-5 spots (mean of 265 vs. 98/1000 responders, p<0.002) and fewer IFN-γ spots (163 vs 386/1000 cells, p<0.005) than MNC-stimulated cells. Further, the ratio of IFN-γ to IL-5 spots was almost ten-fold lower in B cell-stimulated cultures compared to MNC-induced cultures (0.67 vs. 5.2, p<0.001). ELISPOT studies assessing the ratio of IFN-γ to IL-4 spots and ELISA assays comparing IFN-γ and IL-5 levels from culture supernatants demonstrated the same pattern of marked type 2 skewing by B cells. This pattern was unaffected by the presence of anti-IL-4 antibody suggesting type 2 skewing was not mediated by IL-4. Cytokine skewing produced by B cells or MNC could be partially reversed by swapping MNC and B cells during re-stimulation on day 7, but this plasticity was markedly reduced after 3 (weekly) cycles of B cell or MNC re-stimulation in vitro. Type 2 skewing by B cells was enhanced when monocytes were removed from responder T cell populations by either depleting CD14+ positive cells or by positive selection of T cells prior to stimulation. In contrast, type 2 polarization could be prevented using recombinant IL-12. Not all cells of B-cell origin share the same propensity to type 2 skewing observed with IL-4/CD40L-stimulated B cells; under identical conditions, EBV-transformed B cells stimulated alloimmune T cells to produce a strong type 1 cytokine response comparable to that produced by MNCs. In summary, IL-4/CD40L-stimulated B cells strongly promote a type 2 T cell response during primary alloimmune challenge; this skewing can become fixed after repeated B cell stimulation. Investigators using these cells as APC should be aware of this potential phenomenon, particularly during primary T cell responses. It is also important to consider the factors described above that may exacerbate or ameliorate this effect.

Enhancing Cytotoxicity of Autologous T Cells in AML By Blockade of CTLA-4 and PD-1

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4057-4057 ◽  
Author(s):  
Kirsten Marie Boughan ◽  
Xiaohua Chen ◽  
Paul Szabolcs
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Response ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
T Cell Response ◽  
Inhibitory Receptors

Abstract Background: AML remains a disease diagnosed in the aging population with chemotherapy followed by bone marrow transplant in some cases being the standard of care. Although response rates remain around 50-60%, treatment related mortality and disease relapse remain high. Adoptive immunotherapy, especially those targeting T cell co-inhibitory receptors, has proven successful in solid malignancies however, AML remains less explored. Our laboratory has previously demonstrated the feasibility to generate autologous AML reactive T cells in vitro (Mehta/Szabolcs; Immunotherapy 2016). It was noted that "resistant" AML blasts over expressed a number of genes associated with immunosuppressive characteristics. Over expression of these genes may induce T cell functional exhaustion. Therefore, we hypothesized that blocking PD-1 and/or CTLA-4 during co-culture with IFNg activated AML blasts, may enhance T cell activation and cytotoxicity. To test this hypothesis, we tested CTL responses against AML blasts and IFNg ELISpot formation after blocking with PD-1, CTLA-4 or both receptors, and compared the response in untreated T cells. Gene expression profiles of co-stimulatory/co-inhibitory receptors were also monitored to test for correlation. Methods: We evaluated 12 patients with newly diagnosed AML under an IRB approved protocol with written informed consent of patients. Mononuclear cell preparation was generated from fresh marrow samples or drawn from a biorepository of previously cryopreserved leukophereses. T cells were then purified using immunomagnetic CD3/CD28 beads (Life technologies) and cultivated in media with IL-2 and IL-7 for 2 weeks. AML blasts were cultured over a supporting layer of mesenchymal stromal cells (MSCs) derived from healthy BM donors for 1 week and then cryopreserved. T cells were then co-cultured with restored and irradiated autologous AML cells at an effector: target (E: T) ratio of 5:1 to 40:1. AML and T cells were co-cultured in the presence of Ipilimumab (anti-CTLA-4), or Nivolumab (anti-PD-1), or a combination of both drugs. T cells and AML were re stimulated in X-vivo 15 with IL-12, IL-15 and IL-2 weekly x 3weeks. T cell response to AML was quantitated by IFNg ELISpot assay and Europium TDA (EuTDA) CTL assays independently. Co-stimulatory/co-inhibitory expression on T cells was examined with RT-q PCR assay. Paired-sample student t test was used for statistical analysis with p<0.05. Results and Discussion: Out of 12 samples, 10 (83%) yielded viable AML cells available for cytotoxicity assay. One third (33%) of co-cultures exhibited a positive T cell response in CTL assays ("killers"). There was no difference in CTL activity by blockade of either PD-1 or CTL-4 (Fig 1). IFN-ɣ spot formation in ELISpot was observed in 4/10 samples (40%) with statistical significance noted in cells blocked with PD-1 as compared to all other blockade types (Fig 2). The results indicated that in vitro priming with autologous AML blasts or together with blocking PD-1 can enhance T cell response in 33-40%. By gene expression analysis, the ratio of co-stimulatory to co-inhibitory genes was calculated. In PD-1 blocked cells, the ratio of activation/inhibition was not impacted in T cells from "killers" (0.9; p=0.1), however, T cells from "non-killer cells" had a diminished ratio due to higher expression of co-inhibitory molecules (0.4; p=0.04) (Fig 3). This trend was also present in CTLA-4 blocked cells (0.85; p=0.4 in killers vs 0.54; p=0.03 in non-killers) (data not shown). Interestingly, dual blockage failed to influence gene expression ratio, data not shown. Conclusion: The above studies demonstrate that cytotoxicity can be achieved in T cells when primed against autologous AML. PD-1 blockade can enhance IFNg production and cytotoxic responses, but CTLA-4 and dual blockade failed to enhance T cell function. The upregulation of an inhibitory pattern of genes in T cells that did not express cytotoxicity (non-killers) could allude to an "inhibitory phenotype" that may be resistant to immunotherapy drug blockade and requires further study. Disclosures No relevant conflicts of interest to declare.

Whole-tumor-antigen-pulsed dendritic cells elicit cytotoxic T-cell response against pediatric nasopharyngeal carcinoma in vitro

2008 ◽  
Vol 26 (1) ◽  
pp. 78-85 ◽  
Author(s):  
Dou Yufeng ◽  
Zhang Guocheng ◽  
Xu Dongliang ◽  
Fu Rong ◽  
Cao Yuhong ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
T Cell ◽  
Nasopharyngeal Carcinoma ◽  
Tumor Antigen ◽  
Cell Response ◽  
T Cell Response ◽  
Cytotoxic T Cell

scholarly journals Role of viral infectivity in the induction of influenza virus-specific cytotoxic T cells.

1978 ◽  
Vol 147 (4) ◽  
pp. 1236-1252 ◽  
Author(s):  
T J Braciale ◽  
K L Yap
Keyword(s):  
T Cells ◽  
Influenza Virus ◽  
T Cell ◽  
Cell Response ◽  
Infectious Virus ◽  
Cytotoxic T Cells ◽  
T Cell Response ◽  
Cytotoxic T Cell

This report examines the requirement for infectious virus in the induction of influenza virus-specific cytotoxic T cells. Infectious influenza virus was found to be highly efficient at generating both primary and secondary cytotoxic T-cell response in vivo. Inactivated influenza virus however, failed to stimulate a detectable cytotoxic T-cell response in vivo even at immunizing doses 10(5)-10(6)-fold higher than the minimum stimulatory dose of infectious virus. Likewise inactivated virus failed to sensitize target cells for T cell-mediated lysis in vitro but could stimulate a specific cytotoxic response from primed cells in vitro. Possible requirements for the induction of virus-specific cytotoxic T-cell responses are discussed in light of these observations and those of other investigators.

scholarly journals Dysregulated T Cell Response in Bone Marrow Immune Micro-Environment of Patients with Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3140-3140
Author(s):  
Yu-tong Wang ◽  
Yuan Kong ◽  
Yang Song ◽  
Zheng-Fan Jiang ◽  
Xiao-jun Huang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Response ◽  
Graft Function ◽  
T Cell Response ◽  
Intracellular Cytokine ◽  
Hematopoietic Stem ◽  
Poor Graft Function

Abstract Background: Poor graft function (PGF), a kind of bone marrow (BM) failure syndrome, is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nevertheless, the exact mechanisms underlying PGF remain unclear. The BM immune micro-environment is considered to be involved in the regulation of murine hematopoiesis. Dysregulated T cell response was found to suppress proliferation and induce apoptosis of hematopoietic progenitor cells in patients with aplastic anemia. Therefore, we conducted a study to analyze the alteration of T cell subpopulations in BM micro-environment of allotransplant patients. Aims: To compare the cellular compositions and function of T cells in BM micro-environment between patients with PGF and good graft function (GGF) after allo-HSCT in Peking University Institute of Hematology. Methods: Using a prospective nested case-control study, the active phenotype and memory phenotype of CD4+ T cells and CD8+ T cells in BM were analyzed by flow cytometry in 12 patients with PGF, 36 matched patients with GGF after allo-HSCT, and 15 healthy donors (HDs). Furthermore, the cytokine secretion function of CD4+ T cells and CD8+ T cells were evaluated after simulation and the level of eight Th1 and Th2 cytokines in BM plasma were detection by cytometric beads assay. Results: The demographic and clinical characteristics were similar between allo-HSCT patients with PGF and those with GGF. Although the PGF patients presented a significant lymphopenia, a notable increased percentage of activated CD8+ T cells was detected in the BM of PGF patients when compared to that in GGF patients (61.7% versus 35.0%, P =.02). Moreover, the in vitro cytokine stimulated tests demonstrated a significant higher proportion of Tc1 in PGF patients (46.1% versus 20.3% versus 28.4%, P <.005), an elevated percentage of Th1 in PGF compared with HDs (38.5% versus 21.7%, P <.005), a higher percentage of Th2 (4.5% versus 2.1% versus 2.3%, P <.005) and a dramatically decreased percentage of Tc2 in PGF (0.6% versus 2.0% versus 2.0%, P <.0001). Therefore, a significant elevation in the ratio of Th1/Th2 (19.73 versus 7.39 versus 6.91, P <.0001) and Tc1/Tc2 (67.25 versus 10.07 versus 14.57, P <.005) were observed in PGF when compared with those in GGF and HDs. The changes of IFN-gama and IL-4 levels in BM plasma detected by cytometric beads assay were in accordance with the intracellular cytokine results analyzed by flow cytometry. Summary/Conclusion: Both the in vitro intracellular cytokine testing after stimulation and the BM plasma cytokine detection provides evidence that CD4+ and CD8+ T cells were polarized towards a type-1 cytokine response in patients with PGF, suggesting that the dysfunction of T cell response in BM immune micro-environment may hamper the hematopoietic recovery after allo-HSCT. Acknowledgment: Supported by the National Natural Science Foundation of China (grant nos. 81370638&81230013), and the Beijing Municipal Science and Technology Program (grant nos. Z141100000214011& Z151100004015164& Z151100001615020). Disclosures No relevant conflicts of interest to declare.

scholarly journals Dendritic cells transduced by multiply deleted HIV-1 vectors exhibit normal phenotypes and functions and elicit an HIV-specific cytotoxic T-lymphocyte response in vitro

Blood ◽  
2000 ◽  
Vol 96 (4) ◽  
pp. 1327-1333 ◽  
Author(s):  
Andreas Gruber ◽  
June Kan-Mitchell ◽  
Kelli L. Kuhen ◽  
Tetsu Mukai ◽  
Flossie Wong-Staal
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Ex Vivo ◽  
T Cell Response ◽  
Adoptive T Cell Therapy ◽  
Cytotoxic T Cell ◽  
T Cell Therapy ◽  
Hiv 1

Dendritic cells (DCs) genetically modified to continually express and present antigens may be potent physiologic adjuvants for induction of prophylactic or therapeutic immunity. We have previously shown that an env and nef deleted HIV-1 vector (HIV-1ΔEN) pseudotyped with VSV-G transduced monocyte-derived macrophages as well as CD34+ precursors of DCs. Here we extended these findings with HIV-1ΔEN to highly differentiated human DCs derived in culture from circulating monocytes (DCs). In addition, a new vector derived from HIV-1ΔEN but further deleted in its remaining accessory genes vif, vpr, and vpu(HIV-1ΔEN V3) was also tested. Both vectors efficiently transduced DCs. Transduction of DCs did not significantly alter their viability or their immunophenotype when compared with untransduced DCs. Furthermore, the phagocytic potential of immature DCs, as well as their ability to differentiate into mature DCs capable of stimulating T-cell proliferation, was not affected. Finally, DCs transduced by the HIV-1ΔEN vector were able to elicit a primary antiviral cytotoxic T-cell response in autologous CD8 T cells. These results suggest that HIV-1–based vectors expressing viral antigens may be useful for in vivo active immunization as well as ex vivo priming of cytotoxic T cells for adoptive T-cell therapy.

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