Validation of Stereotyped Immunoglobulin Heavy Chain CDR3 Sequences As Candidate Antigens for Immunotherapy of CLL

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1775-1775
Author(s):  
Cristina Maccalli ◽  
Maria Gounari ◽  
Kostas Stamatopoulos ◽  
Federico Caligaris-Cappio ◽  
Giorgio Parmiani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
T Lymphocytes ◽  
Cell Cultures ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Typing ◽  
Class I

Abstract Abstract 1775 The immunoglobulin gene repertoire in CLL is remarkably restricted with greater than 30% of cases carrying quasi-identical (stereotyped)heavy complementarity-determining region 3 (VH CDR3) sequences. Indeed, cases can be clustered into different subsets based on shared, subset-biased motifs within the clonotypic VH CDR3s, with, notably, only a handful of subsets accounting for almost 10% of all CLL. VH CDR3 stereotypes are more frequent in cases with unmutated IGHV genes (U-CLL) who are associated with adverse prognosis. In principle, VH CDR3 stereotypy might allow to exploit these IG motifs as candidate Tumor Associated Antigens (TAA) for targeted immunotherapy of CLL. The aim of our study was to validate as potential TAA subset-specific IG motifs from major CLL subsets, focusing especially on subsets #1 and #2 that are the largest overall and both associated with aggressive clinical course. We have so far identified, by in silico analysis, 1–3 long peptides (15-mer) encompassing the VH CDR3 protein regions of subsets #1, 2, 4, 6, 8, 10 with (i) high binding score to MHC class II molecules and (ii) also containing minimal HLA class I-specific epitopes (HLA-A2, -A3, -A24, DR1, DR7, DR13 that are most frequent in the Caucasian population). Blood lymphocytes from 18 CLL patients were collected and phenotyped by flow cytometry with appropriate antibodies to assess the expression of stimulatory, co-stimulatory and negative regulatory molecules on both T and B cells. In addition, HLA typing of CLL patients was performed to select patients expressing the aforementioned HLA molecules. Overall, 13/18 patients matched the defined HLA class I and/or class II molecules. Negatively purified T cells from 11 CLL patients expressing HLA-A2 and/or DR13 have been then stimulated in vitro with the synthesized peptides of the specific stereotype (subset #1 and 2) in the presence of culture medium containing 5% of human serum plus IL-2 (20 IU/ml) and IL-15 (10 ng/ml). These T lymphocytes were then weekly stimulated with autologous irradiated antigen presenting cells (APC; monocytes, B cells, etc.) pulsed with the peptides. Starting from the third week of culture, the specific recognition of CDR3-derived TAAs and of tumor cells (autologous CLL cells) by the T cell cultures has been assessed by in vitro functional assays (ELISPOT assay). We were able to isolate CDR3- (subsets #1 and #2) and tumor-specific T cells from 5/11 CLL patients. In addition, in 4 selected patients the Ag- and tumor specific T lymphocytes have been expanded in vitro by Rapid Expansion Protocol (REP), based on the stimulation of T cells with allogeneic irradiated PBMCs from healthy donors plus OKT3 and high doses of IL-2. Using this protocol we were able to obtain large numbers (2–10 ×109) of anti-CDR3 T cells in all 4 cases tested, thereby, in principle, achieving the potential to use this protocol for expanding sufficient cells for clinical applications. Interestingly, post-REP T cell cultures showed enrichment (85–90%) of CD3+CD8+ T cells and down-modulation of negative regulatory molecules, such as CTLA-4, as compared to pre-REP in vitro stimulated T cells. These cells could be expanded in vitro for up to 6 weeks without any decay in proliferation. Taken together, these results indicate that stereotyped VH CDR3 peptide sequences can represent candidate antigens to elicit T cell-mediated anti-CLL responses, especially in poor prognosis cases, where therapeutic innovation is more urgently needed. After validation of this protocol in a larger series, our results may provide the proof of principle for the design of new immunotherapy protocols for CLL, including both active vaccination and adoptive cell therapy. Disclosures: No relevant conflicts of interest to declare.

Analysis of Memory T Lymphocytes Activity Following Stimulation with HLA-A24, A1 and Cw4 Restricted 9- and 10-mer Cytomegalovirus pp65 Overlapping Peptides.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2876-2876
Author(s):  
Monica Ghei ◽  
David F. Stroncek ◽  
Maurizio Provenzano
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Immune Therapy ◽  
Hla Class I ◽  
Class I ◽  
Specific Ctls ◽  
Over Time

Abstract In healthy subjects, primary infection with Cytomegalovirus (CMV) is usually mild or asymptomatic and is effectively controlled by the cell-mediated immune response. However, in immune compromised individuals, such as those with AIDS or after bone marrow transplantation, CMV reactivation is associated with significant morbidity until the individual’s immune system is completely reconstituted. One means of preventing post-transplant CMV infection is adoptive immunotherapy using CMV-specific cytotoxic T cells (CTLs) from the transplant donor. Several 9- and 10-mer HLA class I restricted peptides derived from the immune dominant CMV 65 kd matrix phosphoprotein (pp65) have been shown to produce CMV-specific CTLs. Two overlapping HLA-A24 restricted peptides have been specifically described: pp65 341–349 and pp65 341–350. These are 9- and 10-mer peptides that overlap except for the last amino acid phenylalanine (F) at the C-terminus [QYDPVAALF(F)]. Despite their similarity, the ability of these peptides to induce a T cell response has been reported to differ. Although it has been generally accepted that a unique CMV peptide is bound and presented by each separate HLA class I molecule, recent studies suggest that certain peptides are more promiscuous and may be presented by more than one HLA Class I antigen. For example, the 9-mer pp65 341–349 has been shown to stimulate CTLs from both HLA-A24 and Cw4 donors, while the 10-mer pp65 341–350 has been shown to be reactive with both HLA-A24 and A1 donors. The current investigation sought to compare the potency of these two peptides and determine the optimum peptide size for effective CMV adoptive immune therapy. Both peptides were tested for their ability to stimulate CMV-specific CTLs in HLA-A24, HLA-A1, and HLA-Cw4 restriction. In addition, a pp65 16-mer that included the 9- and 10-mers was tested for its ability to reactivate either CD8+ or CD4+ memory T cells. IFN-γ mRNA transcript as well as protein production were measured by in vitro cell culture assays. Peptide stimulations were performed on isolated CD8 and CD4 T lymphocytes by inducing the cells for 3 hours after a 2-week in vitro sensitization. The goal of the investigation was to determine whether both the 9- and the 10-mer peptides maintained high levels of CTL stimulation over time for all HLA restrictions studied. Moreover, it was important to investigate whether stimulation with the 16-mer, followed by restimulation by the two smaller peptides embedded within the larger sequence, led to effective T cell memory immune response. The 9- and 10-mer peptides effectively stimulated CTLs from HLA-A24, HLA-A1, and HLA-Cw4 CMV seropositive donors. Although both 9- and 10-mer were able to maintain high levels of stimulation over time for all restrictions, the 9-mer induced highest responses in cells expressing HLA-A24 (S.I. 4.07–528) or HLA-Cw4 (S.I. 4.15–483) while the 10-mer induced highest responses in cells expressing HLA-A24 (S.I. 3.5–528) or HLA-A1 (S.I. 8.25–615). The 16-mer peptide was also able to stimulate T cells from all HLA-A24, A1 and Cw4 donors (S.I. 6.95, 4.96, 5.02) at levels that are well maintained over time. This data confirmed that both the 9- and the 10-mer peptides are promiscuous and not restricted to a single HLA antigen. These peptides that have the ability to produce CMV-specific CTLs in patients with several different HLA types present a practical advantage over peptides that are restricted only to a single HLA type, and thus are optimal for CMV adoptive immune therapy.

scholarly journals A Novel Strategy for Off-the-Shelf T Cell Therapy Which Evades Allogeneic T Cell and NK Cell Rejection

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1711-1711
Author(s):  
Yong Zhang ◽  
Surbhi Goel ◽  
Aaron Prodeus ◽  
Utsav Jetley ◽  
Yiyang Tan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Hla Class I ◽  
Class Ii ◽  
Class I

Abstract Introduction. Despite the success of autologous chimeric antigen receptor (CAR)-T cells, barriers to a more widespread use of this potentially curative therapy include manufacturing failures and the high cost of individualized production. There is a strong desire for an immediately available cell therapy option; however, development of "off-the-shelf" T cells is challenging. Alloreactive T cells from unrelated donors can cause graft versus host disease (GvHD) for which researchers have successfully used nucleases to reduce expression of the endogenous T cell receptor (TCR) in the allogeneic product. The recognition of allogeneic cells by the host is a complex issue that has not been fully solved to date. Some approaches utilize prolonged immune suppression to avoid immune rejection and increase persistence. Although showing responses in the clinic, this approach carries the risk of infections and the durability of the adoptive T cells is uncertain. Other strategies include deletion of the B2M gene to remove HLA class I molecules and avoid recognition by host CD8 T cells. However, loss of HLA class I sends a "missing-self" signal to natural killer (NK) cells, which readily eliminate B2Mnull T cells. To overcome this, researchers are exploring insertion of the non-polymorphic HLA-E gene, which can provide partial but not full protection from NK cell-mediated lysis. Because activated T cells upregulate HLA class II, rejection by alloreactive CD4 T cells should also be addressed. Methods. Here, we developed an immunologically stealth "off-the-shelf" T cell strategy by leveraging our CRISPR/Cas9 platform and proprietary sequential editing process. To solve the issue of rejection by alloreactive CD4 and CD8 T cells, we knocked out (KO) select HLA class I and class II expression with a sequential editing process. Additionally, we utilize potent TCR-α and -β constant chain (TRAC, TRBC) gRNAs that achieve >99% KO of the endogenous TCR, addressing the risk of GvHD. An AAV-mediated insertion of a CAR or TCR into the TRAC locus is used in parallel with the TRAC KO step to redirect the T cells to tumor targets of interest. Alloreactivity by CD4 and CD8 T cells, NK killing, GvHD induction and T cell function was assessed in vitro and/or in vivo. Results. By knocking out select HLA class I and class II proteins, we were able to avoid host CD4- and CD8-T cell-mediated recognition. Edited T cells were protected from host NK cells, both in vitro and in an in vivo model engrafted with functional human NK cells. TRAC edited donor T cells did not induce GvHD in an immune compromised mouse model over the 90-day evaluation period. Using our proprietary T cell engineering process, we successfully generated allogeneic T cells with sequential KOs and insertion of a tumor-specific TCR or CAR with high yield. Importantly, these allogeneic T cells had comparable functional activity to their autologous T cell counterparts in in vitro assays (tumor cell killing and cytokine release) as well as in vivo tumor models. With a relatively small bank of donors, we can provide an "off-the-shelf" CAR or TCR-T cell solution for a large proportion of the population. Conclusions. We have successfully developed a differentiated "off-the-shelf" approach, which is expected to be safe and cost-effective. It is designed to provide long-term persistence without the need for an immune suppressive regimen. This promising strategy is being applied to our T cell immuno-oncology and autoimmune research candidates. Disclosures Zhang: Intellia Therapeutics: Current Employment. Goel: Intellia Therapeutics: Current Employment. Prodeus: Intellia Therapeutics: Current Employment. Jetley: Intellia Therapeutics: Current Employment. Tan: Intellia Therapeutics: Current Employment. Averill: Intellia Therapeutics: Current Employment. Ranade: Intellia Therapeutics: Current Employment. Balwani: Intellia Therapeutics: Current Employment. Dutta: Intellia Therapeutics: Current Employment. Sharma: Intellia Therapeutics: Current Employment. Venkatesan: Intellia Therapeutics: Current Employment. Liu: Intellia Therapeutics: Current Employment. Roy: Intellia Therapeutics: Current Employment. O′Connell: Intellia Therapeutics: Current Employment. Arredouani: Intellia Therapeutics: Current Employment. Keenan: Intellia Therapeutics: Current Employment. Lescarbeau: Intellia Therapeutics: Current Employment. Schultes: Intellia Therapeutics: Current Employment.

scholarly journals Fas-Independent Apoptosis of Activated T Cells Induced by Antibodies to the HLA Class I α1 Domain

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3629-3639 ◽  
Author(s):  
Laurent Genestier ◽  
Romain Paillot ◽  
Nathalie Bonnefoy-Berard ◽  
Geneviéve Meffre ◽  
Monique Flacher ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Heavy Chain ◽  
Hla Class I ◽  
Class I ◽  
T Cell Proliferation ◽  
Activated T Cells ◽  
Hla Class I Molecules

Abstract In addition to their major function in antigen presentation and natural killer cell activity regulation, HLA class I molecules may modulate T-cell activation and proliferation. Monoclonal antibodies (MoAbs) that recognize distinct epitopes of HLA class I molecules were reported to interfere with T-cell proliferation. We show here that two MoAbs (mouse MoAb90 and rat YTH862) that bind to an epitope of the α1 domain of HLA class I heavy chain induce apoptotic cell death of activated, but not resting, peripheral T lymphocytes. Other reference anti-HLA class I antibodies specific for distinct epitopes of the α1 (B9.12.1), α2 (W6/32), or α3 (TP25.99) domains of the heavy chain decreased T-cell proliferation but had little or no apoptotic effect. Apoptosis shown by DNA fragmentation, phosphatidylserine externalization, and decrease of mitochondrial transmembrane potential was observed whatever the type of T-cell activator. Apoptosis did not result from Fas/Fas-L interaction and distinct though partly overlapping populations of activated T cells were susceptible to Fas– and HLA class I–mediated apoptosis, respectively. Induction of apoptosis did not require HLA class I cross-linking inasmuch as it could be observed with monovalent Fab′ fragments. The data indicate that MoAb90 and YTH862 directed against the α1 domain of HLA class I trigger apoptosis of activated T lymphocytes by a pathway which does not involve Fas-ligand.

scholarly journals Use of synthetic peptides of influenza nucleoprotein to define epitopes recognized by class I-restricted cytotoxic T lymphocytes.

1987 ◽  
Vol 165 (6) ◽  
pp. 1508-1523 ◽  
Author(s):  
J Bastin ◽  
J Rothbard ◽  
J Davey ◽  
I Jones ◽  
A Townsend
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Infected Cell ◽  
Cytotoxic T Lymphocytes ◽  
Synthetic Peptides ◽  
Class Ii ◽  
Cytotoxicity Assay ◽  
Class I ◽  
Class I Mhc

The conserved epitopes of influenza nucleoprotein (NP) recognized by class I MHC-restricted CTL from CBA (H-2k) and C57BL/10 (H-2b) mice have been defined in vitro with synthetic peptides 50-63 and 365-379, respectively. Two Db-restricted clones were described that recognize different epitopes on peptide 365-379. Finally, the recognition of complete NP was shown to be approximately 200-fold less efficient than peptide in the cytotoxicity assay. These phenomena are closely related to results with class II-restricted T cells and they strengthen the hypothesis that influenza proteins are degraded in the infected cell before recognition by class I-restricted CTL.

Immunological relevance of the tumor associated antigen (TAA) COA-1 in colorectal cancer (CRC)

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 13590-13590
Author(s):  
D. C. Corsi ◽  
C. Maccalli ◽  
M. Ciaparrone ◽  
A. F. Scinto ◽  
G. Cucchiara ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Specific Antigen ◽  
Class Ii ◽  
Hla Typing ◽  
Class I ◽  
Mhc Molecules

13590 Background: Immunotherapy (IT) in CRC has often produced discouraging results. COA-1 is a new TAA recognized by CD4+ T cells from peripheral blood (PB) of a CRC pt; its immunogenic epitope is presented on the surface of tumor cells in association with DRβ1*1301 or *0402 HLA class II molecules. Our aim is verifying whether an immune response directed against COA-1 mediated by CD4+ T cells can be isolated from PB of CRC pts. To achieve a more efficient anti-tumor response a recognition of a specific antigen by both the CD4+ and CD8+ lymphocytes should be performed; so different epitopes deriving from the processing of the same antigen should be presented to the immune system in association with both class I and class II MHC molecules. We identified a list of COA-1 derived peptides with the calculated score for the binding to HLA-A2, the more common HLA class I molecule within the Caucasian population. A failure in generating COA-1 specific T cells was observed in stage I-II CRC pts. Methods: From Jan 04 to day PB samples from 36 CRC pts (14 stage III/ 22 stage IV) have been collected and the HLA typing has been performed. Pts. expressing HLA DRbβ*0402, HLA DRβ1*1301 or HLA-A2 have been selected to collect other blood drawns and verifying whether an immune response directed against COA-1 could be isolated from their PB. Results: 4 pts were positive for the expression of DRβ1*1301 and 2 for the expression of DRβ1*0402. PB lymphocytes have been in vitro stimulated with the COA-1 derived epitopes and tumor reactivity has been verified. An immune response directed to COA-1 was detected in the PB of these 6 pts; anti-COA-1 CD4+ T cells were in vitro isolated and their cytotoxicity measured by granzyme B release. 9 pts were positive for the expression of HLA-A2 and we are stimulating the lymphocytes isolated from these pts with 6 selected COA-1 derived peptides binding the HLA-A2. We observed specific CD8+ T cells for 2 peptides in 1 pt. Conclusions: Our data identify COA-1 like an immunogenic antigen that can evoke an anti-tumor immune response CD4+ mediated in CRC; the response correlates with disease progression. Experiments are ongoing to evaluate an immune response mediated by both CD4+ and CD8+ T cells. These results will determine whether COA-1 could be used for future protocols of IT in CRC. No significant financial relationships to disclose.

Double Loading of Dendritic Cell MHC Class I and MHC Class II with an AML Antigen Repertoire Enhances Primary and Secondary T-Cell Responses In Vitro.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2529-2529
Author(s):  
William K. Decker ◽  
Dongxia Xing ◽  
Sufang Li ◽  
Simon N. Robinson ◽  
Hong Yang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Cd8 T Cells ◽  
Class Ii ◽  
T Cell Response ◽  
Class I ◽  
Cross Presentation ◽  
Ifn Γ

Abstract Despite improvements in therapy for acute myelogenous leukemia (AML), a significant percentage of patients still relapse and succumb to their disease. Dendritic cell immunotherapy offers the promise of potentially effective supportive therapy for a variety of neoplastic conditions; and the use of DCs loaded with tumor antigens is now recognized as an important investigational therapy. Though a variety of methods have been used to load DC vaccines, the loading of the MHC class II compartment with tumor lysate has predominated. The priming of a class II-mediated (CD4) T-cell response may be crucial to the success of DC immunotherapy as such a response is likely required for the development of memory CD8+ T-cells. DC cross-presentation is credited with the ability of lysate-loaded DCs to prime both CD4 and CD8 T-cell responses, enabling the generation of CD8+ CTLs without the loading of the MHC class I compartment (i.e. the cytoplasm). Recently, however, several reports have raised doubts as to the efficiency of cross-presentation as a mechanism for CTL priming in vivo. To examine this issue, we have loaded human DCs with both AML tumor lysate and mRNA. This technique allows the full repertoire of class I antigens to be presented without dependence upon cross-presentation; and, moreover, provides a full complement of class II antigens necessary for CD4 T-cell priming and the generation of memory responses. Methods: CD14+ precursors were isolated from normal donor PBPCs by magnetic separation. Immature DCs were then generated by culturing precursors for six days in GM-CSF and IL-4. Lysate was produced by three successive freeze/thaw cycles of blasts. mRNA was extracted from blasts using Trizol and oligo-dT separation. Immature DCs were pulsed for three hours with AML lysate and subsequently electroporated with AML mRNA. Loaded DCs were matured for 48 hours with IL-1β, TNF-α, IL-6, and PGE2 and then used to prime autologous T-cells. Short-term responses were assayed on day 5 of the 1st stimulation. Memory responses were assayed on day 10 of a tertiary stimulation. Results: Doubly-loaded DCs can prime a superior T-cell response in vitro in comparison to that of singly-loaded DCs, demonstrating a 30–70% increase in IFN-γ ELISpots over lysate-loaded DCs (p<0.001) and a 3–4 fold increase in ELISpots in comparison to mRNA loaded DCs (p<0.001). These results were verified by flow cytometry which showed 35% of CD8+ T-cells primed by doubly-loaded DCs were CD69+/IFN-γ+ vs. 14% of CD8+ T-cells primed by lysate-loaded DCs (p<0.001). This enhancement may be based upon both an upregulation of CD83 surface expression (p<0.0019) of doubly-loaded DCs and/or the upregulation of B7.1/B7.2 that accompanies elevated CD40L signaling. Memory responses were also greatly improved, with a 126% increase in total ELISpots (double loaded DCs versus lysate loaded DCs; p<0.03) and a 187% increase in total IFN-γ secretion (p<0.03). Unloaded (p<0.01) and mRNA (p<0.007) loaded DCs exhibited a virtual inability to generate memory T-cells in vitro, suggesting that the perpetuation of the memory response is reliant upon T-cell help. Conclusion: DCs doubly-loaded with lysate and mRNA are more efficient in the generation of primary and secondary immune responses than are singly-loaded DCs. The clinical administration of such doubly-loaded DCs may offer an important therapeutic option to patients with AML.

Identification of Four New HLA Class II Restricted Minor Histocompatibility Antigens Contributing to Graft Versus Leukemia Reactivity.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3247-3247
Author(s):  
Anita N. Stumpf ◽  
Edith D. van der Meijden ◽  
Cornelis A.M. van Bergen ◽  
Roelof Willemze ◽  
J.H. Frederik Falkenburg ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Cd4 T Cell ◽  
Class I ◽  
T Cell Clones ◽  
Cell Clones

Abstract Patients with relapsed hematological malignancies after HLA-matched hematopoietic stem cell transplantation (HSCT) can be effectively treated with donor lymphocyte infusion (DLI). Donor-derived T cells mediate beneficial graft-versus-leukemia (GvL) effect but may also induce detrimental graft-versus-host disease (GvHD). These T cell responses are directed against polymorphic peptides which differ between patient and donor due to single nucleotide polymorphisms (SNPs). These so called minor histocompatibility antigens (mHag) are presented by HLA class I or II, thereby activating CD8+ and CD4+ T cells, respectively. Although a broad range of different HLA class I restricted mHags have been identified, we only recently characterized the first autosomal HLA class II restricted mHag phosphatidylinositol 4-kinase type 2 beta (LB-PI4K2B-1S; PNAS, 2008, 105 (10), p.3837). As HLA class II is predominantly expressed on hematopoietic cells, CD4+ T cells may selectively confer GvL effect without GvHD. Here, we present the molecular identification of four new autosomal HLA class II restricted mHags recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia (CML) after HLAmatched HSCT who experienced long-term complete remission after DLI with only mild GvHD of the skin. By sorting activated CD4+ T cells from bone marrow mononuclear cells obtained 5 weeks after DLI, 17 highly reactive mHag specific CD4+ T cell clones were isolated. Nine of these T cell clones recognized the previously described HLADQ restricted mHag LB-PI4K2B-1S. The eight remaining T cell clones were shown to exhibit five different new specificities. To determine the recognized T cell epitopes, we used our recently described recombinant bacteria cDNA library. This method proved to be extremely efficient, since four out of five different specificities could be identified as new HLA-class II restricted autosomal mHags. The newly identified mHags were restricted by different HLA-DR molecules of the patient. Two mHags were restricted by HLA-DRB1 and were found to be encoded by the methylene-tetrahydrofolate dehydrogenase 1 (LBMTHFD1- 1Q; DRB1*0301) and lymphocyte antigen 75 (LB-LY75-1K; DRB1*1301) genes. An HLA-DRB3*0101 restricted mHag was identified as LB-PTK2B-1T, which is encoded by the protein tyrosine kinase 2 beta gene. The fourth mHag LB-MR1-1R was restricted by HLA-DRB3*0202 and encoded by the major histocompatibility complex, class I related gene. All newly identified HLA class II restricted mHags exhibit high population frequencies of 25% (LB-MR1-1R), 33% (LB-LY75-1K), 68% (LB-MTHFD1- 1Q), and 70% (LB-PTK2B-1T) and the genes encoding these mHags show selective (LY- 75) or predominant (MR1, MTHFD1, PTK2B) expression in cells of hematopoietic origin as determined by public microarray databases. All T cell clones directed against the newly identified mHags recognized high HLA class II-expressing B-cells, mature dendritic cells (DC) and in vitro cultured leukemic cells with antigen-presenting phenotype. The clone recognizing LB-MTHFD1-1Q also showed direct recognition of CD34+ CML precursor cells from the patient. In conclusion, we molecularly characterized the specificity of the CD4+ T cell response in a patient with CML after HLA-matched HSCT who went into long-term complete remission after DLI. By screening a recombinant bacteria cDNA library, four new different CD4+ T cell specificities were characterized. Our screening method and results open the possibility to identify the role of CD4+ T cells in human GvL and GvHD, and to explore the use of hematopoiesis- and HLA class II-restricted mHag specific T cells in the treatment of hematological malignancies.

scholarly journals Epstein–Barr Virus+ B Cells in Breast Cancer Immune Response: A Case Report

2021 ◽  
Vol 12 ◽  
Author(s):  
Andrea Aran ◽  
Vicente Peg ◽  
Rosa Maria Rabanal ◽  
Cristina Bernadó ◽  
Esther Zamora ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Class Ii ◽  
Hla Typing ◽  
Presentation Pathway ◽  
Tcr Repertoire

EBV-specific T cells have been recently described to be involved in fatal encephalitis and myocarditis in cancer patients after immune checkpoint therapies. Here, we report the study of a human triple-negative breast cancer tumor (TNBC) and EBV-transformed B cells obtained from a patient-derived xenograft (PDX) that progressed into a lymphocytic neoplasm named xenograft-associated B-cell lymphoma (XABCL). T-cell receptor (TCR) high-throughput sequencing was performed to monitor the T-cell clonotypes present in the different samples. Forty-three T-cell clonotypes were found infiltrating the XABCL tissue after three passes in mice along 6 months. Eighteen of these (42%) were also found in the TNBC biopsy. TCR infiltrating the XABCL tissue showed a very restricted T-cell repertoire as compared with the biopsy-infiltrating T cells. Consequently, T cells derived from the TNBC biopsy were expanded in the presence of the B-cell line obtained from the XABCL (XABCL-LCL), after which the TCR repertoire obtained was again very restricted, i.e., only certain clonotypes were selected by the B cells. A number of these TCRs had previously been reported as sequences involved in infection, cancer, and/or autoimmunity. We then analyzed the immunopeptidome from the XABCL-LCL, to identify putative B-cell-associated peptides that might have been expanding these T cells. The HLA class I and class II-associated peptides from XABCL-LCL were then compared with published repertoires from LCL of different HLA typing. Proteins from the antigen processing and presentation pathway remained significantly enriched in the XABCL-LCL repertoire. Interestingly, some class II-presented peptides were derived from cancer-related proteins. These results suggest that bystander tumor-infiltrating EBV+ B cells acting as APC may be able to interact with tumor-infiltrating T cells and influence the TCR repertoire in the tumor site.

scholarly journals Comprehensive analysis of T cell immunodominance and immunoprevalence of SARS-CoV-2 epitopes in COVID-19 cases

2020 ◽  
Author(s):  
Alison Tarke ◽  
John Sidney ◽  
Conner K Kidd ◽  
Jennifer M. Dan ◽  
Sydney I. Ramirez ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Responses ◽  
Hla Class I ◽  
Class Ii ◽  
Comprehensive Analysis ◽  
Class I ◽  
Entire Genome ◽  
Cell Responses ◽  
Global Coverage

SUMMARYT cells are involved in control of SARS-CoV-2 infection. To establish the patterns of immunodominance of different SARS-CoV-2 antigens, and precisely measure virus-specific CD4+ and CD8+ T cells, we studied epitope-specific T cell responses of approximately 100 convalescent COVID-19 cases. The SARS-CoV-2 proteome was probed using 1,925 peptides spanning the entire genome, ensuring an unbiased coverage of HLA alleles for class II responses. For HLA class I, we studied an additional 5,600 predicted binding epitopes for 28 prominent HLA class I alleles, accounting for wide global coverage. We identified several hundred HLA-restricted SARS-CoV-2-derived epitopes. Distinct patterns of immunodominance were observed, which differed for CD4+ T cells, CD8+ T cells, and antibodies. The class I and class II epitopes were combined into new epitope megapools to facilitate identification and quantification of SARS-CoV-2-specific CD4+ and CD8+ T cells.

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