Restoration of tumor specific human leukocyte antigens class I-restricted cytotoxicity by dendritic cell stimulation of tumor infiltrating lymphocytes in patients with advanced ovarian cancer

2004 ◽  
Vol 14 (1) ◽  
pp. 64-75 ◽  
Author(s):  
A. D. Santin ◽  
S. Bellone ◽  
M. Palmieri ◽  
B. Bossini ◽  
S. Cane' ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Tumor Antigen ◽  
Advanced Ovarian Cancer ◽  
Human Leukocyte ◽  
Hla Class I ◽  
Class I ◽  
Autologous Tumor

Despite the large number of potentially cytotoxic tumor-infiltrating (TIL) and tumor-associated (TAL) lymphocytes accumulated in the peritoneal cavity ascitic fluid and tumor tissue, advanced ovarian cancer is a progressive disease, suggesting that TIL and TAL populations eventually become functionally suppressed in vivo. Dendritic cells (DC) are the most powerful professional antigen presenting cells known in humans and recently, ovarian tumor antigen pulsed DC have been shown to elicit tumor specific human leukocyte antigens (HLA)-class I-restricted cytotoxicity from the peripheral blood of advanced ovarian cancer patients. In this study, we have evaluated the potential of tumor antigen-pulsed fully mature DC stimulation in restoring tumor-specific cytotoxicity in anergic TIL populations from advanced ovarian cancer patients. In addition, we have compared tumor-specific T-cell responses induced by tumor antigen-loaded DC in TIL to those induced in TAL and peripheral blood lymphocytes (PBL). DC stimulation induced powerful cytotoxicity against autologous tumor target cells in TIL-derived CD8+ T-cells from all patients tested, while autologous Epstein–Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) were not lysed. Killing of autologous tumor cells was higher by CD8+ T-cells from TIL compared to PBL and TAL (P < 0.01) and was more strongly inhibited by anti-HLA class I MAb (P < 0.05 compared to PBL and TAL). Phenotypically, all cytotoxic T lymphocyte (CTL) populations were CD3+/CD8+, with variable levels of CD56 expression. Finally, although a marked Type 1 cytokine bias [ie, interferon-gamma/interleukin-4 (IFN-γhigh/IL-4low)] was observable in all DC-stimulated CD8+ T-cell populations, TIL derived CD8+ T-cells showed a higher percentage of IFN-γ positive cells compared to TAL and PBL. Taken together, these data show that tumor lysate-pulsed DC can consistently restore strong CD8+ CTL responses from TIL against autologous ovarian cancer cells. DC-stimulated TIL may represent a superior source of tumor-specific CTL for adoptive T-cell immunotherapy for advanced ovarian cancer.

scholarly journals MHC class I-presented tumor antigen appraisable for T-cell responses against ovarian cancer

2015 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Jing Yao Wang ◽  
Nan Zhang ◽  
Xiaojie Yang ◽  
Danli Gao ◽  
Lirong Yin ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
T Cell ◽  
Mhc Class I ◽  
Tumor Antigen ◽  
Human Leukocyte ◽  
T Cell Responses ◽  
Mean Value ◽  
Class I ◽  
Cell Responses

<p>The purpose of this study is to assess whether MHC class I-presented tumor antigen is appraisable for T-cell responses against ovarian cancer. In ovarian cancer cell, human leukocyte antigen A2 (HLA-A2) associated with peptides was used to promote the activation of naive T cells so as to activate antigen-specific T cells. 7 or 4 patients were observed grade 1 or 2 injection site reactions, respectively. 5, 2 or 1 patients were observed grade 1, 2 or 3 pain reactions, respectively. 4 or 1 patients were observed grade 1 or 2 induration reactions. Total number mean value of patients experiencing response to the particular peptide was 7.73, and total number mean value of peptides to which the patients responded was 7.45. MHC class I-presented tumor antigen is appraisable for T-cell responses against ovarian cancer in China.</p>

scholarly journals HLA Class I-T Cell Epitopes from trans-Sialidase Proteins Reveal Functionally Distinct Subsets of CD8+ T Cells in Chronic Chagas Disease

2008 ◽  
Vol 2 (9) ◽  
pp. e288 ◽  
Author(s):  
María G. Alvarez ◽  
Miriam Postan ◽  
D. Brent Weatherly ◽  
María C. Albareda ◽  
John Sidney ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chagas Disease ◽  
Cd8 T Cells ◽  
Hla Class I ◽  
Class I ◽  
T Cell Epitopes ◽  
Chronic Chagas Disease

Redirected CD4+ T Cells towards HLA Class I-Restricted WT1 Epitope Successfully Display Multifactorial Helper Function for the Enhancement of Antileukemia Efficacy Mediated by Redirected T-Cell Based Immunocelltherapy.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3153-3153
Author(s):  
Yukihiro Miyazaki ◽  
Hiroshi Fujiwara ◽  
Toshiki Ochi ◽  
Sachiko Okamoto ◽  
Hiroaki Asai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Hla Class I ◽  
Class I ◽  
Leukemia Cells ◽  
Proliferation And Differentiation ◽  
Ifn Γ

Abstract Abstract 3153 Purpose: In antitumor adoptive immunotherapy, the utility of tumoricidal CD8+ T cells are mainly highlighted, while in tumor immunity, the importance of tumor-reactive CD4+ T cells is also well documented. However, because the number of well-characterized tumor-associated epitopes recognized by CD4+ T cells still remains small, application of tumor-reactive CD4+ T cells is limited. In order to circumvent this drawback, redirection of CD4+ T cells to well-characterized HLA class I-restricted CD8+ T-cell epitope seems promising. In this study, using an HLA class I-restricted and WT1-specific T-cell receptor (TCR) gene transfer, we, in detail, examined helper functions mediated by those gene-modified CD4+T cells in redirected T cell-based antileukemia adoptive immunotherapy. Methods: HLA-A*2402-restricted and WT1235–243-specific TCR α/β genes were inserted into our unique retroviral vector encoding shRNAs for endogenous TCRs (WT1-siTCR vector), and was employed for gene-modification both of CD4+ and CD8+ T cells to express WT1-specific TCR. (1) WT1 epitope-responsive cytokine production mediated by WT1-siTCR-transduced CD4+ T cells (WT1-siTCR/CD4) was measured using bead-based immunoassay and ELISA assay. (2) WT1 epitope-ligation induced co-stimulatory molecules by WT1-siTCR/CD4 was assessed using flow cytometry. (3) Impacts on WT1 epitope and leukemia-specific responses; cytocidal activity, proliferation and differentiation into memory T-cell phenotype, mediated by WT1-siTCR-transduced CD8+ T cells (WT1-siTCR/CD8) provided by concurrent WT1-siTCR/CD4 were assessed using 51Cr-release assay, CD107a/intracellular IFN-γ assay, CFSE dilution assay and flow cytometry. (4) WT1 epitope-ligation triggered chemokine production mediated by WT1-siTCR/CD4 was assessed using real-time PCR, then chemotaxis mediated by WT1-siTCR/CD8 in response to those chemokines was assessed using a transwell experiment. (5) In vivo tumor trafficking mediated by WT1-siTCR/CD4 was assessed using bioluminescence imaging assay. (6) Finally, WT1-siTCR/CD4-caused in vivo augmentation of antileukemia functionality mediated by WT1-siTCR/CD8 was assessed similarly using a xenografted mouse model. Results: WT1-siTCR/CD4 showed a terminal effector phenotype; positive for transcription factor T-bet, but negative for Bcl-6 or Foxp3. Upon recognition of WT1 epitope, WT1-siTCR/CD4 produced Th1, but not Th2 cytokines in the context of HLA-A*2402, which simultaneously required HLA class II molecules on target cells. WT1 epitope-ligation enhanced WT1-siTCR/CD4 to express cell-surface OX40. In the presence of WT1-siTCR/CD4, but not non-gene-modified CD4, effector functions mediated by WT1-siTCR/CD8 in response to WT1 epitope and leukemia cells, including cytocidal activity based on CD107a expression and IFN-γ production was enhanced. Such augmentation was mediated by humoral factors produced by WT1 epitope-ligated WT1-siTCR/CD4. Additionally, proliferation and differentiation into memory phenotype, notably CD45RA- CD62L+ central memory phenotype, mediated by WT1-siTCR/CD8 in response to both WT1 epitope and leukemia cells were also augmented, accompanied with increased expression of intracellular Bcl-2 and cell-surface IL-7R. Next, CCL3/4 produced by activated WT1-siTCR/CD4 triggered chemotaxis of WT1-siTCR/CD8 which express the corresponding receptor, CCR5. Using bioluminescence imaging, intravenously infused WT1-siTCR/CD4 successfully migrated towards leukemia cells inoculated in a NOG mouse. Finally, co-infused WT1-siTCR/CD4 successfully augmented immediate accumulation towards leukemia cells and antileukemia reactivity mediated by WT1-siTCR/CD8 in a xenografted mouse model. Conclusion: Using GMP grade WT1-siTCR vector, redirected CD4+ T cells to HLA class I-restricted WT1 epitope successfully recognized leukemia cells and augmented in vivo antileukemia functionality mediated by similarly redirected CD8+ T cells, encompassing tumor trafficking, cytocidal activity, proliferation and differentiation into memory cells. The latter seem to support the longevity of transferred antileukemia efficacy. Taking together, coinfusion of redirected CD4+ T cells to HLA class I-restricted WT1 epitope seems feasible and advantageous for the successful WT1-targeting redirected T cell-based immunotherapy against human leukemia. Disclosures: No relevant conflicts of interest to declare.

Generation of Cytotoxic T Cell Responses Directed to HLA Class I Restricted Epitopes from the Aspergillus f16 Allergen.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1644-1644
Author(s):  
Gamal Ramadan ◽  
Barbara Davies ◽  
Viswanath P. Kurup ◽  
Carolyn A. Keever-Taylor
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Response ◽  
T Cell Responses ◽  
Peptide Pool ◽  
Hla Class I ◽  
T Cell Response ◽  
Class I ◽  
Cell Responses

Abstract Invasive pulmonary aspergillosis is a primary cause of morbidity and mortality in immunocompromised patients such as hematopoietic progenitor cell transplant patients. Studies both in patients with allergic bronchopulmonary aspergillosis and murine models demonstrated the importance of a CD4+ Th1 T cell response in conferring protection from infection or preventing disease progression. The role of CD8+ T cell response to A. fumigatus is less clear. Our efforts to develop effective immunotherapeutic approaches against A. fumigatus included preparation of 104 overlapping pentadecapeptides spanning the 427 aa coding region of the aspergillus allergen, Asp f16 previously shown to induce T cell responses. Each 15 aa peptide overlaps the preceding peptide by 11 aa. Monocytes from healthy donors were treated with GM-CSF and IL-4 for 2-3 days to generate immature dendritic cells (fast DC), pulsed with a pool containing 1 μg of each pentadecapeptide, then matured with inflammatory cytokines (IL-1β, IL-6, PGE2 and TNF-alpha) for 2 days. Mature, pulsed fast DC were used to prime proliferative and CTL responses (weekly primings). T cells from 5/5 donors proliferated to the peptide pool. CTL lines were obtained from each of the first two donors that were primed. After 4 weeks the line from donor #2 was strongly cytotoxic to autologous peptide pool-pulsed and aspergillus culture extract-pulsed DC and peptide pool pulsed HLA Class I matched BLCL. Supernatant from this line killed fresh aspergillus conidia. Six of 21 smaller pools of 4-11 peptides showed reactivity. Specificity could be narrowed by screening peptides shared by the pools to 3 candidate peptides. Pool-pulsed BLCL matched for only 1 or 2 HLA alleles were used to demonstrate CTL restriction by HLA-B-3501. A database search of peptides likely to be restricted to B3501 identified the likely sequences as YFKYTAAAL, LPLCSAQTW, and GTRFPQTPM. Each induced similar reactivity when pulsed onto B-3501+ targets. CD8+ T cells steadily increased from 5.2% at week 3 to 19.0% after the 7th priming. CTL activity and IFNγ production were exclusively mediated by CD8+ T cells and CD107a was expressed by 42% of the CD8+ T cells in response to pool-pulsed BLCL indicating degranulation. CTL cross-reacted with pool pulsed B3503+ BLCL but not B3502+, or B3508+ BLCL. B3503+ BLCL presented YFKYTAAAL and to a lesser extent GTRFPQTPM but not peptide LPLCSAQTW. Our data show that DC pulsed with a pentadecapeptide pool from Asp f16 are capable of inducing a CD8+, HLA-Class I restricted Aspergillus-specific T cell response directed to multiple peptides contained within the pool. Further characterization of this system is in progress to identify additional immunogenic peptides from Asp f16 that might be useful in clinical immunotherapy protocols to prime protective immune responses to prevent or treat aspergillus infection.

4-1BB (CD137) or CD40 Signaling Fails To Improve the Expansion of Antigen Specific T Cells Demonstrated with Engagement of TCR, CD28 and CD83 Ligand.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2665-2665 ◽  
Author(s):  
Naoto Hirano ◽  
Marcus O. Butler ◽  
Lee M. Nadler
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Hla Class I ◽  
Cd40 Ligand ◽  
Cd4 T Cell ◽  
Class I ◽  
Stimulatory Capacity ◽  
Costimulatory Signals ◽  
The Impact

Abstract Following the engagement of the T cell receptor by HLA class I and antigenic peptide, naïve CD8+ T cells are primed to receive one or more costimulatory signals. Some of these signals, which are upregulated on and delivered by mature dendritic cells, include members of the immunoglobulin superfamily such as CD80 and CD83. Using a K562 derived artificial antigen presenting cell (aAPC) that expresses HLA-A2, CD80, and CD83, we have shown that the coengagement of CD83 ligand:CD83 and CD28:CD80 induces prolonged and preferential expansion of antigen specific CD8+ T cells. Furthermore, we have found that CD28:CD80 signaling is required for the induction of CD83 ligand expression on peripheral T cells. In order to identify additional immunoaccessory molecules that can augment this response, we have developed a system to efficiently transfer any chosen molecule into aAPC. This provides an excellent platform for studying a potentially immunogenic molecule given the relative lack of immunoaccessory molecules expressed by K562 (i.e. no expression of CD40, CD40 ligand, CD83, CD86, 4-1BB, 4-1BB ligand, OX40, OX40 ligand, HLA class I, or HLA class II). Following the transduction of a candidate molecule under study, the stimulatory capacity of a supertransduced aAPC can be compared to parental aAPC. Attractive candidates include members of the TNF superfamily since they have been shown to deliver important costimulatory signals to T cells. It has been suggested that 4-1BB signaling supports the survival of newly generated effector CD8+ T cells and that CD40 signaling confers “CD4+ T cell-like” help directly to CD8+ T cells. However, the impact of each of these molecules on the stimulation and expansion of antigen specific T cells has not been exhaustively studied. In this report, we transfected aAPC with either 4-1BB ligand or CD40 ligand, allowing us to compare the stimulatory capacity of aAPC/CD40 ligand, aAPC/4-1BB ligand and parental aAPC. We stimulated HLA-A2 positive CD8+ T cells from healthy donors three times at weekly intervals with A2-restricted MART1 peptide pulsed onto either irradiated aAPC/CD40 ligand, aAPC/4-1BB ligand or parental aAPC. Between the stimulations, cells were treated with IL2 and IL15 every three days. When MART1 peptide pulsed aAPC/CD40 ligand were used as stimulators, the total number of CD8+ T cells and number of MART1 specific CD8+ T cells was slightly smaller. IFN-γ ELISPOT analysis revealed that functional avidity of T cell receptors on MART1 specific CD8+ T cells was similar whether they were stimulated by aAPC/CD40 ligand or parental aAPC. These results indicate that CD40 ligand, at least in the human setting, does not directly provide “CD4+ T cell-like” help to antigen-specific CD8+ T cells. In contrast, stimulation with peptide pulsed aAPC/4-1BB ligand did generate a larger total number of CD8+ T cells. Surprisingly, however, most of these T cells were not antigen specific. In fact, significantly fewer MART1 specific CD8+ T cells were generated by aAPC/4-1BB ligand compared to aAPC alone. These results suggest that, unlike CD80 and CD83, 4-1BB ligand delivers a costimulatory signal resulting in the non-specific expansion of CD8+ T cells. This work demonstrates the versatility of our system to dissect the function of particular immunoaccessory molecules and determine the optimal conditions in the stimulation and expansion of antigen-specific human CD8+ T cells ex vivo.

Extracellular Domains of CD8a and β Subunits Are Required and Sufficient for HLA Class I Restricted Helper Activity of TCR-Engineered CD4+ T Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3574-3574
Author(s):  
Marleen M van Loenen ◽  
Renate S. Hagedoorn ◽  
Roelof Willemze ◽  
J.H. Frederik Falkenburg ◽  
Mirjam H.M. Heemskerk
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Hla Class I ◽  
Extracellular Domain ◽  
Class I ◽  
Α Subunit ◽  
Extracellular Domains ◽  
Helper Activity

Abstract Abstract 3574 Poster Board III-511 Adoptive transfer of T cell receptor (TCR)-transferred T cells may be an attractive strategy to treat patients with hematological malignancies relapsing after allogeneic stem cell transplantation. Transfer of HLA class I restricted TCRs into CD8+ T cells demonstrated redirected antigen specificity. However, for persistence of anti-leukemic responses in vivo, CD4+ T cells may be important. Therefore, redirecting specificity of CD4+ T cells with well defined HLA class I restricted TCRs might be an attractive strategy for providing help. HLA class I restricted TCRs mostly are CD8-dependent, so for optimal HLA class I restricted reactivity, it was demonstrated that co-expression of the CD8-coreceptor is necessary. The CD8 molecule is expressed on the T cell surface as an αα or an αβ dimer. The α subunit of the CD8 coreceptor binds to the non-polymorphic residues in the α3 domain of the HLA class I molecules thereby enhancing the avidity of the TCR/MHC complex, and the cytoplasmatic tail of the α subunit directly associates with the protein tyrosine kinase Lck (p56lck), promoting signal transduction after T cell activation. The β subunit of the CD8 coreceptor is able to strengthen the avidity of the CD8/MHC/TCR interaction via its extracellular domain, and the intracellular domain enhances the association with the intracellular molecules p56Lck and LAT. Previously, it was reported that for optimal HLA class I restricted specific reactivity with respect to proliferation, cytokine production and cytotoxicity, co-expression of the CD8αβ; coreceptor was needed whereas co-expression of the CD8αα coreceptor marginally increased HLA class I restricted functional activity. Since the regulation of the introduced TCR as well as the CD8 coreceptor in redirected CD4+ T cells will be mediated by retroviral LTRs, we prefer to co-transfer a signaling deficient CD8 coreceptor, thereby minimizing the risk of overstimulation of the redirected T cells. In this study, we investigated whether co-transfer of a signaling deficient CD8 coreceptor would still result in optimal HLA class I restricted functionality of HLA class I restricted TCR engineered CD4+ T cells. For this purpose, we constructed retroviral constructs encoding either wild type CD8α or CD8β subunits, CD8α subunits in which the LCK binding domain was mutated, CD8 subunits composed of the CD8α extracellular domain coupled to the intracellular CD8β signalling domain, and intracellular truncated CD8α or CD8β subunits. pp65-KYQ specific CD4+ T cells were isolated using the IFNγ capture assay and transduced with HLA class I restricted TCRs. Subsequently, TCR transduced virus specific CD4+ T cells were sorted based on marker gene expression, and transduced with the different CD8α and CD8β combinations. In agreement with previous studies we demonstrate that for optimal helper activity of the HLA class I restricted TCR transferred CD4+ T cells coexpression of the CD8αβ coreceptor was required. T cells produced IFNγ, TNFα and IL-2, upregulated CD40L and proliferated upon antigen specific stimulation of the HLA class I restricted TCR. Truncation of the intracellular domains of the CD8α and CD8β subunits did not change the functionality of the HLA class I restricted TCR transferred CD4+ T cells. Whereas in the thymus both the intracellular and extracellular domains of CD8β contribute independently to positive selection and development of CD8+ T cells, our results demonstrate that for optimal HLA class I restricted functionality of TCR modified virus specific CD4+ T cells only the extracellular domains of the CD8a and β subunits are required and sufficient. Disclosures: No relevant conflicts of interest to declare.

Co-Administration Of Gene-Modified CD4+ T Cells Targeting HLA Class I-Restricted WT1 Epitope Diversely Enhances The Antitumor Effect Mediated By Redirected T-Cell Based Adoptive Immunotherapy Against Human Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 144-144
Author(s):  
Hiroshi Fujiwara ◽  
Fumihiro Ochi ◽  
Toshiki Ochi ◽  
Hiroaki Asai ◽  
Yukihiro Miyazaki ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
Cd8 T Cells ◽  
Adoptive Immunotherapy ◽  
Cd4 T Cells ◽  
Hla Class I ◽  
Class I ◽  
Leukemia Cells

Abstract Purpose In the context of redirected T-cell based antitumor adoptive immunotherapy, the therapeutic roles played by co-infused CD4+ T cells genetically redirected to the predefined HLA class I-restricted epitope which had been originally recognized by effector CD8+ T cells has not yet been fully discussed. In this study, using an HLA class I-restricted WT1 -specific T-cell receptor (TCR) gene transfer, we in detail examined antileukemia functionality mediated by these gene-modified CD4+ T cells co-infused with similarly gene-modified effector CD8+ T cells as the redirected T cell-based adoptive immunotherapy. Methods Using our unique retroviral vector expressing HLA-A*2402-restricted and WT1235-243-specific TCR a/b genes and shRNAs for endogenous TCRs (WT1-siTCR vector), we genetically modified both CD4+ and CD8+ T cells from the same healthy donor or leukemia patients (termed WT1-siTCR/CD4 and WT1-siTCR/CD8, respectively). First, target-responsive cellular outputs mediated by WT1-siTCR/CD4 was thoroughly examined using flowcytometry, ELISA, 51Cr-release assay, CFSE dilution assay and bioluminescence assay. Next we similarly assessed impacts of WT1-siTCR/CD4 on the antileukemia functionality mediated by concurrentWT1-siTCR/CD8 both in vitro and in vivo. Eventually, we assessed the in vivo therapeutic efficacy of combined administration of WT1-siTCR/CD8 with WT1-siTCR/CD4 using a xenografted mouse model. Results The transcription factor profile demonstrated that WT1-siTCR/CD4 turned a terminal effector, but not regulatory phenotype. Activated WT1-siTCR/CD4 expressed cell-surface CD40L. Target-responsive cytokine production profile of WT1-siTCR/CD4 represented the Th1 helper function in the context of HLA-A*2402. HLA class II molecules expressed by leukemia cells facilitated the recognition of leukemia cells by WT1-siTCR/CD4 in the context of HLA-A*2402. WT1-siTCR/CD4 displayed the delayed cytocidal activity determined by 51Cr release assay. WT1-siTCR/CD4 could produce IFN-g in response to freshly isolated leukemia cells. WT1-siTCR/CD4 displayed the leukemia trafficking activity in vivo. WT1-siTCR/CD4 represented the potential to migrate into bone marrow via CXCR4/CXCL12 axis both in vitro and in vivo. Concurrent WT1-siTCR/CD4 augmented IFN-g production and cytotoxic degranulation mediated by WT1-siTCR/CD8 in response to the cognate epitope via humoral factors. Consequently, the cytocidal activity against autologous leukemia cells mediated by WT1-siTCR/CD8 was augmented in the presence of WT1-siTCR/CD4, both of them generated from normal lymphocytes of the same patient with leukemia in a complete remission. Upon the target recognition, activated WT1-siTCR/CD4 recruited WT1-siTCR/CD8 via CCL3/4-CCR5 axis. Proliferative response and differentiation into central memory T-cell subset mediated by WT1-siTCR/CD8 in response to the cognate epitope and leukemia cells were enhanced in the presence of autologousWT1-siTCR/CD4, but not gene-modified CD4+ T cells (NGM-CD4). CD127 expression on activated WT1-siTCR/CD8 also increased in parallel to this differentiation. Co-infused WT1-siTCR/CD4 augmented the tumor trafficking and persistence of WT1-siTCR/CD8 in vivo, resulting in the greater suppression of leukemia cells in a xenografted mouse model. Finally, in the therapeutic mouse model, co-infusion of WT1-siTCR/CD8 with of WT1-siTCR/CD4 significantly suppressed the growth of inoculated leukemia cells compared to that in mice received co-infusion of WT1-siTCR/CD8 with NGM-CD4 (Fig.1). Correlation between the therapeutic efficacy and survival of infused gene-modified T cells was also observed. Conclusion In results, the combined infusion of WT1-siTCR/CD8 with WT1-siTCR/CD4, but not NGM-CD4 obviously demonstrates the enhanced antileukemia efficacy via diverse mechanisms. Now we have just started a clinical trial using gene-modified T cells with WT1-siTCR vector for the treatment of patients with refractory acute myeloid leukemia and myeloid dysplastic syndrome. Because redirected T cells employed in this trial encompassed both WT1-siTCR/CD4 and WT1-siTCR/CD8, we are planning to clinically verify the significance of WT1-siTCR/CD4 in the redirected T cell-based antileukemia adoptive immunotherapy. (Fig.1) Disclosures: No relevant conflicts of interest to declare.

Characterization of T Cell Epitopes of the Receptor for Hyaluronic Acid Mediated Motility (RHAMM/CD168) in Acute Myeloid Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2540-2540
Author(s):  
Michael Schmitt ◽  
Li Li ◽  
Mark Ringhoffer ◽  
Thomas Barth ◽  
Markus Wiesneth ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Hyaluronic Acid ◽  
T Cell ◽  
Cd8 T Cells ◽  
Myeloid Leukemia ◽  
Hla Class I ◽  
Class I ◽  
T Cell Epitopes ◽  
Acute Myeloid

Abstract To improve the clinical outcome of patients with acute myeloid leukemia (AML), immune therapies targeting leukemia associated antigens (LAAs) might be an approach complementary to chemotherapy and transplantation of hematopoetic stem cells. The receptor for hyaluronic acid mediated motility (RHAMM/CD168) has been defined as a LAA with specific expression. To define T cell epitopes of RHAMM/CD168 towards specific T cell immunotherapies, ten peptides were synthesized considering different computer algorithms and subjected to ELISPOT assays for interferon gamma and granzyme B, and to Cr-51 release assays. CD8+ T cells taken from the peripheral blood (PB) of 13 AML patients and presensitized with the RHAMM/CD168-derived peptides R3 (ILSLELMKL) or R5 (SLEENIVIL) did specifically recognize T2 cells pulsed with R3/R5. In contrast, CD8+ T cells isolated from the PB of 21 healthy volunteers were not able to lyse R3 or R5 pulsed T2 cells, even after presensitization. COS7 cells co-transfected with HLA-A*0201 and RHAMM/CD168 were lysed by R3 or R5 presensitized CD8+ T cells. Single HLA-A*0201 or RHAMM/CD168 transfected COS7 were not recognized. Cross-reactivity of the T cells was excluded by the use of unrelated peptides. K562 cells positive for RHAMM/CD168, but lacking HLA-class I molecules were not recognized indicating T cells and not NK cells as effector cells. The HLA class-I restricted lysis of COS-7 HLA-A*0201 and RHAMM/CD168 double- transfectants was confirmed by HLA class-I blocking antibody experiments. In an AML patient having received AML blast-derived dendritic cells, a higher frequency of RHAMM/CD168-peptide specific T cells was observed after four vaccinations when compared to his T cell status before vaccination. RHAMM/CD168 is also expressed in patients with other hematological malignancies which suggests a broad clinical applicability of its newly characterized T cell epitope peptides as a potential cancer vaccine.

Identification and Characterization of HLA Class I-Restricted MYD88 L265P-Derived Peptides As Tumor-Specific Targets for Immunotherapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2750-2750
Author(s):  
Annika Nelde ◽  
Juliane S Stickel ◽  
Daniel Johannes Kowalewski ◽  
Oliver Olaf Wolz ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Hla Class I ◽  
Class I ◽  
Specific Cell ◽  
Specific Lysis ◽  
Effector Cells

Abstract Non-Hodgkin lymphomas (NHL) are frequent malignancies with considerable mortality. A recurrent somatic and oncogenic driver mutation in the Toll-like receptor adaptor gene MYD88, Leu265Pro (L265P) has been identified in up to 90% of certain NHL subtypes. Genetic alterations affecting a protein-coding region have the potential to generate mutation-derived peptides that are presented by HLA class I proteins and might be recognized by cytotoxic T cells. Because MYD88L265P is a widely occurring and tumor-specific mutation, we investigated the potential of MYD88L265P -containing peptides for CD8+ T cell mediated immunotherapy as a new therapeutic approach for MYD88L265P+ NHL. Based on in silico prediction we identified potential HLA ligands encompassing the MYD88L265P mutation for several HLA class I allotypes. Functional characterization of the candidate HLA class I MYD88L265P-derived HLA class I ligands with regard to induction of T cell responses identified a set of immunogenic peptides for HLA-B*07 and -B*15. In one MYD88L265P-mutated NHL patient, memory T cell responses targeting three different MYD88L265P-derived HLA class I ligands were detected by IFN-γ ELISPOT. Efficient T cell priming was demonstrated in vitro using naïve T cells of healthy volunteers (HVs). In detail, three HLA-B*07 peptides (P1-3B*07) and one HLA-B*15-restricted peptide (P4B*15) were analyzed using artificial antigen-presenting cell-based (aAPC) in vitro priming experiments in three to six HVs, respectively. For all tested peptides proliferation of peptide-specific CD8+ T cells could be detected after in vitro priming. For the HLA-B*07-restricted ligands, peptide-specific CD8+ T cells could be induced in 6/6 (P1B*15), 1/3 (P2B*07) and 3/4 (P3B*07) HVs, respectively, with a maximum frequency of 14.1% peptide-specific CD8+ T cells. For the HLA-B*15-restricted ligand (P4B*15), peptide-specific CD8+ T cells could be induced in 2/3 HVs with a maximum frequency of 9.5% tetramer-positive CD8+ T cells. The functionality and specificity of peptide-specific CD8+ T cells after aAPC-based in vitro priming was validated by intracellular cytokine staining for IFN-γ and TNF-α as well as for the expression of the degranulation marker CD107a. In 3/3 HVs primed with P1B*07 (RPIPIKYKAM) as well as in 1/2 HVs primed with P4B*15 (HQKRPIPIKY), we detected specific and functional CD8+ T cell populations after stimulation with the mutated peptides, but not after stimulation with the corresponding wild type peptides (P1WT: RLIPIKYKAM, P4WT: HQKRLIPIKY). Furthermore, the peptide-specific cytotoxic activity of specific CD8+ T cells was demonstrated in a VITAL assay. The polyclonal P1B*07- and P4B*15-specific CD8+ T cells (0.12% and 0.76% peptide-specific T cells, respectively) lysed autologous peripheral blood mononuclear cells loaded with the mutated peptides, but not cells presenting the wild type peptides. P4B*15-specific CD8+ T cells showed 17.9% (±1.2%) MYD88L265P-peptide-specific cell killing at an E/T ratio of 1:1 compared to 2.6% (±1.2%) of non-specific cell lysis of unspecific effector cells against the same targets in three independent replicates, respectively. The specific lysis showed an E/T ratio-dependent manner as the specific lysis decreases with reducing E/T ratios. P1B*07-specific CD8+ T cells specifically killed 11.4% (±1.7%) of MYD88L265P loaded targets at an E/T ratio of 0.7:1 in comparison to 2.1% unspecific lysis of unspecific effector cells. In this study, we identified and characterized MYD88L265P mutation-derived HLA class I ligands for T cell mediated immunotherapy. The strong immunogenicity of the HLA-B*07 and HLA-B*15-restricted mutation-derived peptides as well as the functionality and specificity of peptide-specific CD8+ T cells, demonstrated by cytotoxicity assays, underline the potential of the MYD88L265P mutation as tumor-specific target. These data highlight the potential of MYD88L265P mutation-specific immunotherapy as a novel broadly applicable and tumor-specific treatment approach for patients with MYD88L265P+ NHL. Disclosures Langerak: InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics.; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam).

scholarly journals 4473 Immune control of plasma cell disorders – in-depth analysis of Sox2 immunity in MGUS

2020 ◽  
Vol 4 (s1) ◽  
pp. 136-136
Author(s):  
Sandra Patricia Susanibar Adaniya ◽  
Casey L. Cummins ◽  
Miren L. Baroja ◽  
Beatriz Carreno ◽  
Gerald P. Linette ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Hla Class I ◽  
Cross Sectional Study ◽  
Class I ◽  
Cell Reactivity ◽  
Cross Sectional ◽  
Depth Analysis

OBJECTIVES/GOALS: We aim to identify and characterize anti-Sox2-specific CD8+ T cell responses in stable MUGS patients expressing HLA class I alleles-A*02:01 and /or -B*07:02. METHODS/STUDY POPULATION: Cross sectional study of patients with stable MGUS defined as stable serum paraprotein for ≥ 12 months from the MM Research Clinic at the Abramson Cancer Institute. Sox2 T cell reactivity will be assessed by IFN-γ ELISPOT assays. Rested PBMC will be pulsed with candidate Sox2-derived peptides predicted to display high affinity to HLA class I alleles and known to be processed and presented as determined by “targeted MS/MS” (mass spectrometry). The presence of anti-Sox2-specific CD8+ T cells will be confirmed in peptide/HLA multimer assays using flow cytometry. Anti-Sox2-specific CD8+ T cells will be characterized for HLA restriction and TCR αβ composition. RESULTS/ANTICIPATED RESULTS: Our work is still in progress. From Aug to Dec 2019, 22 MGUS subjects have been analyzed, 11 of which were found to have the HLA of interest. Positive Sox-2 reactivity by ELISpot was found in 3 subjects. DISCUSSION/SIGNIFICANCE OF IMPACT: Anti-Sox2 immune responses may maintain MGUS in a clinical indolent state by eliminating Sox2-expressing clonogenic MM cells. A detailed characterization of anti-Sox2 T cells followed by in-vivo assessment of their anti-myeloma activity could provide the foundation for a Sox2 based immunotherapy approach in MM.

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