342: Obliterative Airway Disease (OAD) Induced by Antibodies to MHC-Class I Is B Cell Dependent: An Obligatory Role for Antibodies to Self Antigens in the Pathogenesis of OAD

Abstract Abstract 2838 To circumvent cytotoxic T lymphocyte (CTL) tolerance of tumour-associated antigens, the concept of redirecting CTLs against non-cognate targets has developed. One way of doing this is to use bispecific antibodies comprising anti-CD3 and anti-tumour antigen moieties. Unfortunately, this is frequently associated with unacceptable toxicity due to inflammatory cytokine release. As an alternative our approach has been to use a bivalent conjugate recognising a tumour antigen (through an antibody fragment) and a defined population of CTLs (specific for a single antigenic peptide e.g. viral epitope) through peptide presented in the context of recombinant MHC class I. We have produced a conjugate consisting of an anti-human CD20 Fab' fragment joined via a chemical crosslinker (succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate) to murine MHC class I/peptide (Kbα1-α3 domains/β2microglobulin presenting the ovalbumin-derived peptide SIINFEKL; expressed bacterially as a continuous polypeptide single chain trimer after Yu et al, J Immunol 2002). Size exclusion chromatography allowed purification of conjugates with [Fab':MHC class I/peptide] ratios of 1:1 and 2:1 (F2 and F3 respectively). In vitro both constructs were able to redirect the transgenic murine CTL line OT-1 (specific for KbSIINFEKL) to lyse human CD20+ tumour cells (lymphoblastoid Daudi cell line) at effector: target ratios of 10:1. This lysis could be blocked by the addition of 100 fold excess of either anti-CD20 F(ab')2 or the Kb/SIINFEKL-specific antibody 25D1. The constructs were also able to cause in vitro proliferation of naïve OT-1 cells (but not irrelevant CD8+ T cells) in the presence of human CD20+ cells in both thymidine incorporation and CFSE dilution assays. Using a human CD20 transgenic mouse model (Ahuja et al, J Immunol 2007) we have evaluated both constructs in vivo for their ability to redirect adoptively transferred OT-1 cells to deplete B cells from the peripheral blood. A single dose of 1 nmole F3 and 2 nmole F2 caused respectively up to 95% and 85% B cell depletion at day 7. The efficacy of lower doses suggested a dose: response relationship. As a marker of toxicity, we have measured cytokine levels at 2, 8 and 24 hours following a dose of 1 nmole F3 and compared them to those seen after administration of an [anti-CD3 × anti-CD20] bispecific F(ab')2 at a dose (0.5 nmole) which produced similar day 7 peripheral blood B cell depletion: phosphate-buffered saline was given as a negative control. Maximal cytokine release was seen at 2 hours with the levels of IL-4, IL-5, KC, IL-2 and IL-10 being lower after administration of the F3 than after the bispecific F(ab')2. However, interestingly, the F3 resulted in greater IL-12 release. Overall these data suggest that [Fab' × MHC class I/peptide] constructs have the potential to redirect non-cognate CTLs to deplete CD20+ malignant B cells from the peripheral blood and that this is associated with a lower level of cytokine release than a similarly efficacious dose of an anti-CD3-containing bispecific F(ab')2. Furthermore, the ability of [Fab' × MHC class I/peptide] constructs to cause proliferation of OT-1 cells in vitro suggests it may be possible to use a single molecule to both generate a secondary cytotoxic T cell response and subsequently to retarget it, increasing the viability of the approach if adopted in the clinic. Disclosures: No relevant conflicts of interest to declare.

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Potential immune epitope map for structural proteins of SARS-CoV-2

10.21203/rs.3.rs-47663/v1 ◽

2020 ◽

Author(s):

Yengkhom Damayanti Devi ◽

Himanshu Ballav Goswami ◽

Sushmita Konwar ◽

Chandrima Doley ◽

Anutee Dolley ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Mhc Class I ◽

Mhc Class Ii ◽

Cell Epitope ◽

Class I ◽

Structural Proteins ◽

S Protein ◽

N Protein ◽

B Cell Epitope

Abstract Researchers around the world are developing more than 145 vaccines (DNA/mRNA/whole-virus/viral-vector/protein-based/repurposed vaccine) against the SARS-CoV-2 and 21 vaccines are in human trials. However, a limited information is available about which SARS-CoV-2 proteins are recognized by human B- and T-cell immune responses. Using a comprehensive computational prediction algorithm and stringent selection criteria, we have predicted and identified potent B- and T-cell epitopes in the structural proteins of SARS-CoV and SARS-CoV-2. The amino acid residues spanning the predicted linear B-cell epitope in the RBD of S protein (370-NSASFSTFKCYGVSPTKLNDLCFTNV-395) have recently been identified for interaction with the CR3022, a previously described neutralizing antibody known to neutralize SARS-CoV-2 through binding to the RBD of the S protein. Intriguingly, most of the amino acid residues spanning the predicted B-cell epitope (aa 331-NITNLCPFGEVFNATRFASVYAWNRK-356, 403-RGDEVRQIAPGQTGKIADYNYKLPD-427 and aa 437- NSNNLDSKVGGNYNYLYRLFRKSNL-461) of the S protein have been experimentally verified to interact with the cross-neutralizing mAbs (S309 and CB6) in an ACE2 receptor-S protein interaction independent-manner. In addition, we found that computationally predicted epitope of S protein (370-395) is likely to function as both linear B-cell and MHC class II epitope. Similarly, 403-27 and 437-461 peptides of S protein were predicted as linear B cell and MHC class I epitope while, 177-196 and 1253-1273 peptides of S protein were predicted as linear and conformational B cell epitope. We found MHC class I epitope 316-GMSRIGMEV-324 predicted as high afﬁnity epitope (HLA-A*02:03, HLA-A*02:01, HLA-A*02:06) common to N protein of both SARS-CoV-2 and SARS-CoV (N317-325) was previously shown to induce interferon-gamma (IFN-γ) in PBMCs of SARS-recovered patients. Interestingly, two MHC class I epitopes, 1041-GVVFLHVTY-1049 (HLA-A*11:01, HLA-A*68:01, HLA-A*03:01) and 1202-FIAGLIAIV-1210 (HLA-A*02:06, HLA-A*68:02) derived from SARS-CoV S protein with epitope conservancy between 85 to 100% with S protein of SARS-CoV-2 was experimentally verified using PBMCs derived from SARS-CoV patients. We observed that HLA-A*02:01, HLA-A*02:03, HLA-A*02:06, HLA-A*11:01, HLA-A*30:01, HLA-A*68:01, HLA-A*68:02, HLA-B*15:01 and HLA-B*35:01 have been predicted to bind to the maximum number of MHC class I epitope (based on the criterion of allele predicted to bind more than 30 epitopes) of S protein of SARS-CoV-2. Similarly, we observed that HLA-A*02:06, HLA-A*30:01, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*68:01, HLA-A*68:02, HLA-B*15:01 and HLA-B*35:01 are predicted to bind to the maximum number of MHC class I epitope of N protein of SARS-CoV-2. We found that HLA-DRB1*04:01, HLA-DRB1*04:05, HLA-DRB1*13:02, HLA-DRB1*15:01, HLA-DRB3*01:01, HLA-DRB3*02:02, HLA-DRB4*01:01, HLA-DRB5*01:01, HLA-DQA1*04:01, DQB1*04:02, HLA-DPA1*02:01, DPB1*01:01, HLA-DPA1*01:03, DPB1*02:01, HLA-DPA1*01:03, DPB1*04:01, HLA-DPA1*03:01, DPB1*04:02, HLA-DPA1*02:01, DPB1*05:01, HLA-DPA1*02:01, and DPB1*14:01 are predicted to bind to the maximum number of MHC class II epitope of S protein of SARS-CoV-2. Alleles such as HLA-DRB1*04:01, HLA-DRB1*07:01, HLA-DRB1*08:02, HLA-DRB1*09:01, HLA-DRB1*11:01, HLA-DRB1*13:02, HLA-DRB3*02:02, HLA-DRB5*01:01, HLA-DQA1*01:02, DQB1*06:02, DPB1*05:01 and HLA-DPA1*02:01 are found to interact with the maximum number of MHC class II epitope of N protein of SARS-CoV-2. Using the IEDB tool we found the occurrence of HLA alleles with population coverage of around 99% throughout the world. The findings of computational predictions of mega-pool of B- and T-cell epitopes identified in the four main structural proteins of SARS-CoV-2 provides a platform for future experimental validations and the results of present works support the use of RBD or the full-length S and N proteins in an effort towards designing of recombinant protein-based vaccine and a serological diagnostic assay for SARS-CoV-2.

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Characterization of Cellular Immune Responses to a Recombinant Idiotype Vaccine by ELISPOT and Identification of MHC Class I-Restricted T Cell Epitopes by Peptide Mapping.

Blood ◽

10.1182/blood.v104.11.1409.1409 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1409-1409 ◽

Cited By ~ 1

Author(s):

Cristina M. Bertinetti ◽

Hendrik Veelken

Keyword(s):

T Cell ◽

Immune Responses ◽

B Cell ◽

Mhc Class I ◽

B Cell Lymphoma ◽

Class I ◽

Fab Fragment ◽

Cellular Immune Responses ◽

Idiotype Vaccine ◽

Cellular Immune

Abstract Induction of tumor-specific immune responses by idiotype vaccination is a promising strategy for biological therapy of indolent B cell lymphomas. In a previous report, we have described immune responses in a subset of patients participating in a phase I clinical trial primarily designed to demonstrate safety and efficacy of a recombinant idiotype vaccine (Veelken et al., ASH abstract #3342, 2003). In this trial, B-NHL patients who had relapsed after standard chemotherapy received repetitive intradermal vaccinations with recombinant idiotype Fab fragment derived from their tumor mixed with lipid-based adjuvant and concurrent subcutaneous GM-CSF at the same site. We now present the final analysis of cellular immune responses in this cohort. Peripheral blood lymphocytes (PBL) were obtained prior to and on various time points during and after vaccinations. Cryopreserved PBL were stimulated twice by autologous dendritic cells (DC) exposed to the autologous Fab protein for cross-presentation as MHC class I-bound peptides. INFγ-secreting cells were subsequently quantified by ELISPOT with Fab-presenting DC. Alternatively, freshly thawed PBL were directly assayed with recombinant Fab by ELISPOT without prestimulation. An increase in the frequency of Fab-responding PBL was detected in 7 of 15 evaluable patients with the prestimulation assay and in 4 of 10 patients by direct quantitation, resulting in a combined cellular response rate of 53% (9 of 17). A cellular immune response showed a trend for correlation with extended progression-free survival (p=0.08). T cell responses were predominantly idiotype-specific since lesser or no increases in IFNγ-secreting cells were detected against light chain- and VH family-matched control Fabs. Interestingly, a much higher base-line reactivity was observed against the control Fabs in comparison to the patient’s lymphoma Fab in four patients, pointing to the possibility of tumor-specific anergy in lymphoma patients that can at least be partially corrected by active immunization. In an effort to identify the MHC class I-presented idiotype-derived peptides, potential binding motifs were defined by reverse immunology with the SYFPEITHI algorithm (www.syfpeithi.de). Ten candidate peptides from the variable and constant region of an immune responder’s idiotype heavy chain were synthesized and evaluated with post-vaccination PBL by ELISPOT without prestimulation. A peptide derived from the CDR2 region showed a significantly higher response compared to an unrelated peptide control (p=0.0013). Additional peptides derived from the FWR1, CDR1, and CDR2 also showed a significant stimulation, but only in comparison to a no peptide control. ELISPOT offers a valuable tool to monitor cellular immune reponses and demonstrates successful induction of tumor immunity in pretreated, tumor bearing and immunosuppressed B cell lymphoma patients. Supported by Deutsche Krebshilfe

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Genes and Proteins Associated with the Tumor Microenvironment Are Differentially Expressed in Cured Versus Primary Chemotherapy-Refractory Diffuse Large B-Cell Lymphoma.

Blood ◽

10.1182/blood.v110.11.3171.3171 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3171-3171

Author(s):

Johan Linderoth ◽

Patrik Edén ◽

Mats Ehinger ◽

Jeanette Valcich ◽

Mats Jerkeman ◽

...

Keyword(s):

T Cells ◽

Tumor Microenvironment ◽

B Cell ◽

Mhc Class I ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Differentially Expressed ◽

Class I ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Background: The aim of this study was to identify possible differences in gene expression profiles (GEP) between two clinically very different patient populations, cured versus primary chemotherapy-refractory diffuse large B-cell lymphoma (DLBCL). Material and methods: Tumor samples from 37 patients with de novo DLBCL, stage II-IV, either in continuous primary complete remission (n=24) or with progressive disease during primary treatment (n=13) were selected from a larger study cohort and examined with respect to GEP using spotted 55K oligonucleotide arrays. Formalin fixed paraffin-embedded tissue for immunohistochemical analysis was available in 33 of 37 cases, and was used for confirmation on protein level. Results: The top 86 genes with the greatest statistical ability of discriminating the two patient groups were chosen for further analysis. Seventy-nine were over-expressed in the cured cohort, many of them coding for proteins expressed in the tumor microenvironment: CD68, several proteolytic enzymes and proteins involved in remodelling of extra cellular matrix and inflammation. Furthermore, MHC class I molecules, CD3δ, NK transcript 4, and ICAM-1 were overexpressed indicating an enhanced immunological reaction. Immunohistochemistry showed that the frequency of cells expressing CD68, defining the macrophage population, was higher in the cured cohort and that expression of lysozyme, cathepsin D, UPAR, and ICAM-1, was mainly seen within the reactive cells. Tumor infiltrating CD8+ T-cells were more frequent within the cured cohort, corresponding to the increased MHC class I expression seen within this group. Conclusions: In DLBCL, genes coding for antigens present in the tumor microenvironment are differentially expressed in patients with cured vs. chemotherapy-refractory disease. Our findings suggest that a reactive microenvironment, including tumor infiltrating T-cells and macrophages, may have impact on outcome of chemotherapy in DLBCL.

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Novel epitope based peptides for vaccine against SARS-CoV-2 virus: immunoinformatics with docking approach

International Journal of Research in Medical Sciences ◽

10.18203/2320-6012.ijrms20202875 ◽

2020 ◽

Vol 8 (7) ◽

pp. 2385 ◽

Cited By ~ 1

Author(s):

Jesvin Bency B. ◽

Mary Helen P. A.

Keyword(s):

B Cell ◽

Mhc Class I ◽

Mhc Class Ii ◽

Peptide Vaccine ◽

Human Leukocyte ◽

Respiratory Illness ◽

Class Ii ◽

Class I ◽

T Cell Epitopes ◽

B Cell Epitopes

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative viral strain for the contagious pandemic respiratory illness in humans which is a public health emergency of international concern. There is a desperate need for vaccines and antiviral strategies to combat the rapid spread of SARS-CoV-2 infection.Methods: The present study based on computational methods has identified novel conserved cytotoxic T-lymphocyte epitopes as well as linear and discontinuous B-cell epitopes on the SARS-CoV-2 spike (S) protein. The predicted MHC class I and class II binding peptides were further checked for their antigenic scores, allergenicity, toxicity, digesting enzymes and mutation.Results: A total of fourteen linear B-cell epitopes where GQSKRVDFC displayed the highest antigenicity-score and sixteen highly antigenic 100% conserved T-cell epitopes including the most potential vaccine candidates MHC class-I peptide KIADYNYKL and MHC class-II peptide VVFLHVTYV were identified. Furthermore, the potential peptide QGFSALEPL with high antigenicity score attached to larger number of human leukocyte antigen alleles. Docking analyses of the allele HLA-B*5201 predicted to be immunogenic to several of the selected epitopes revealed that the peptides engaged in strong binding with the HLA-B*5201 allele.Conclusions: Collectively, this research provides novel candidates for epitope-based peptide vaccine design against SARS-CoV-2 infection.

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Constitutive Expression of Interleukin (IL)-4 In Vivo Causes Autoimmune-type Disorders in Mice

Journal of Experimental Medicine ◽

10.1084/jem.185.2.329 ◽

1997 ◽

Vol 185 (2) ◽

pp. 329-340 ◽

Cited By ~ 110

Author(s):

Klaus J. Erb ◽

Beate Rüger ◽

Maja von Brevern ◽

Bernhard Ryffel ◽

Annelise Schimpl ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Mhc Class I ◽

Mhc Class Ii ◽

Neutralizing Antibodies ◽

Ex Vivo ◽

Constitutive Expression ◽

Class I

The transgenic (tg) expression of interleukin (IL)-4 under the control of a major histocompatibility complex (MHC) class I promoter leads to B cell hyperactivity in mice, characterized by increased B cell surface MHC class II and CD23 expression, elevated responsiveness of the B cells to polyclonal ex vivo stimulation, and increased immunoglobulin (Ig)G1 and IgE serum levels. Tg mice develop anemia, glomerulonephritis with complement and immune deposition in the glomeruli, and show increased production of autoantibodies. Treatment of IL-4 tg mice with anti-IL-4 neutralizing antibodies protected the mice from disease development, showing that IL-4 was responsible for the observed disorders. Deletion of superantigen responsive autoreactive T cells in the IL-4 tg mice was normal and treatment of mutant mice with deleting anti-CD4 antibodies failed to ablate the onset of autoimmune-like disease, suggesting that CD4+T cells were not the primary cause of the disorders. Furthermore, the deletion of B cells reacting against MHC class I molecules was also normal in the IL-4 tg mice. Therefore the most likely explanation for the increased production of autoantibodies and the autoimmunelike disorders is that IL-4 acts directly on autoreactive B cells by expanding them in a polyclonal manner. Taken together our results show that inappropriate multi-organ expression of IL-4 in vivo leads to autoimmune-type disease in mice.

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