Interleukin 2 Production by Alloantigen- Stimulated CD4+ and CD8+ Human T Cell Subsets: Frequency of HLA Class I or Class II - Reactive Precursor Cells and Clonal Specificity of Activated T Cells

Immunobiology ◽  
1989 ◽  
Vol 179 (4-5) ◽  
pp. 366-381 ◽  
Author(s):  
Jutta Jooss ◽  
Thomas H. Eiermann ◽  
Hermann Wagner ◽  
Dieter Kabelitzi
Keyword(s):  
T Cells ◽  
T Cell ◽  
Interleukin 2 ◽  
Hla Class I ◽  
Class Ii ◽  
Precursor Cells ◽  
T Cell Subsets ◽  
Class I ◽  
Activated T Cells ◽  
Human T Cell

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 Greatly reduced lymphoproliferation in lpr mice lacking major histocompatibility complex class I.

1995 ◽  
Vol 181 (2) ◽  
pp. 641-648 ◽  
Author(s):  
M A Maldonado ◽  
R A Eisenberg ◽  
E Roper ◽  
P L Cohen ◽  
B L Kotzin
Keyword(s):  
T Cells ◽  
Major Histocompatibility Complex ◽  
T Cell ◽  
Class Ii ◽  
T Cell Subsets ◽  
Cell Surface Receptor ◽  
Class I ◽  
Major Histocompatibility ◽  
Gamma Delta ◽  
Histocompatibility Complex

Mice homozygous for the lpr gene have a defect in fas (CD95), a cell surface receptor that belongs to the tumor necrosis factor receptor family and that mediates apoptosis. This genetic abnormality results in lymphoproliferation characterized by the accumulation of CD4-CD8- (double negative [DN]) T cells, autoantibody production, and background strain-dependent, end-organ disease. Our previous results suggested that major histocompatibility complex (MHC) class I may be involved in the development of DN cells. To test this hypothesis, we derived C57BL/6-lpr/lpr (B6/lpr) mice that were deficient for the beta 2-microglobulin gene (beta 2m lpr) and had no detectable class I expression. At 6 mo of age, compared with B6/lpr littermates with normal class I genes, these mice showed greatly reduced lymphadenopathy, mostly due to a dramatic decrease in the number of DN cells. Significant changes in the percentage of other T cell subsets were noted, but only gamma/delta+ T cells showed a marked increase in both percentage and absolute numbers. Analysis of T cell receptor V beta expression of the remaining DN T cells in beta 2m -lpr mice showed a shift to a CD4-like repertoire from a CD8-like repertoire in control B6/lpr mice, indicating that a small MHC class II selected DN population was unmasked in lpr mice lacking class I. We also found that the production of immunoglobulin G (IgG) autoantibodies (antichromatin and anti-single stranded DNA), total IgG and IgG2a, but not total IgM or IgM rheumatoid factor, was significantly reduced in the beta 2m -lpr mice. This work suggests that >90% of DN T cells in lpr mice are derived from the CD8 lineage and are selected on class I. However, a T cell subset selected on class II and T cells expressing gamma/delta are also affected by the lpr defect and become minor components of the aberrant DN population.

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 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.

HLAs, TCRs, and KIRs, a Triumvirate of Human Cell-Mediated Immunity

2020 ◽  
Vol 89 (1) ◽  
pp. 717-739 ◽  
Author(s):  
Zakia Djaoud ◽  
Peter Parham
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
T Cell Receptors ◽  
Hla Class I ◽  
Class Ii ◽  
Class I ◽  
Antigenic Specificity ◽  
Class I Mhc ◽  
Cell Receptors

In all human cells, human leukocyte antigen (HLA) class I glycoproteins assemble with a peptide and take it to the cell surface for surveillance by lymphocytes. These include natural killer (NK) cells and γδ T cells of innate immunity and αβ T cells of adaptive immunity. In healthy cells, the presented peptides derive from human proteins, to which lymphocytes are tolerant. In pathogen-infected cells, HLA class I expression is perturbed. Reduced HLA class I expression is detected by KIR and CD94:NKG2A receptors of NK cells. Almost any change in peptide presentation can be detected by αβ CD8+ T cells. In responding to extracellular pathogens, HLA class II glycoproteins, expressed by specialized antigen-presenting cells, present peptides to αβ CD4+ T cells. In comparison to the families of major histocompatibility complex (MHC) class I, MHC class II and αβ T cell receptors, the antigenic specificity of the γδ T cell receptors is incompletely understood.

Regulation of MHC class II expression in human T-cell malignancies

Blood ◽  
2004 ◽  
Vol 103 (4) ◽  
pp. 1438-1444 ◽  
Author(s):  
Tjadine M. Holling ◽  
Erik Schooten ◽  
Anton W. Langerak ◽  
Peter J. van den Elsen
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Mhc Class Ii ◽  
Lymphoblastic Leukemia ◽  
Class Ii ◽  
Activated T Cells ◽  
Prolymphocytic Leukemia ◽  
Human T Cell ◽  
T Cell Lines

Abstract Expression of major histocompatibility complex (MHC) class II molecules in human activated T cells is under normal circumstances regulated exclusively by the CIITA-PIII subtype of the class II transactivator (CIITA). In this study, we show that the absence of MHC class II expression in leukemic T cells was due to a lack of expression of CIITA, whereas in T-lymphoma cells, expression of CIITA correlated with expression of MHC class II. Interestingly, activation of a CIITA-promoter (P)III–reporter construct was not affected in leukemic T cells. This revealed that the absence of endogenous CIITA expression was not caused by a lack of transcription factors critical for CIITA-PIII activation but suggests the involvement of an epigenetic silencing mechanism. Subsequent analysis showed that the lack of human leukocyte antigen–DR (HLA-DR) expression correlated with hypermethylation of CIITA-PIII in leukemic T-cell lines and in primary T-cell acute lymphoblastic leukemia (T-ALL) and a T-cell prolymphocytic leukemia (T-PLL). Treatment of leukemic T-cell lines with a demethylation agent showed re-expression of CIITA-PIII and HLA-DRA. Furthermore, in vitro methylation of CIITA-PIII and subsequent assessment of CIITA-PIII activity in Jurkat leukemic T cells resulted in reduction of constitutive and CREB-1 (cyclic adenosine monophosphate [cAMP]–response element binding protein 1)–induced promoter activity. Together, these results argue for an important role of DNA hyper-methylation in the control of CIITA expression in leukemic T cells.

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 The HLA Ligandome Landscape of Chronic Myeloid Leukemia Delineates Novel T-Cell Epitopes for Immunotherapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4243-4243
Author(s):  
Tatjana Bilich ◽  
Annika Nelde ◽  
Leon Bichmann ◽  
Helmut R. Salih ◽  
Daniel Johannes Kowalewski ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Cell Responses ◽  
Tki Treatment

Abstract Chronic myeloid leukemia (CML) is characterized by the translocation t(9;22), which leads to the formation of the BCR-ABL fusion transcript. Several approved tyrosine kinase inhibitors (TKIs) target the resulting fusion protein, leading to an improved prognosis of CML patients. Currently, the main treatment goal is the achievement of a deep molecular response (MR), in which TKI therapy can be terminated. Several studies provide evidence that immunological control plays a major role for the course of CML and contributes to the achievement of deep MR in CML patients under TKI treatment (CMLTKI). This implies that reinforcing these immune responses might sustain long-term TKI-free survival or even cure for CML patients. Besides unspecific immunotherapy, such as interferon or immune checkpoint blocking antibodies, a more specific and minor side effect targeting of CML cells might be achieved by antigen-specific immunotherapy approaches. The prerequisite for such strategies is the identification of T-cell targets represented by tumor-associated human leukocyte antigen (HLA)-presented peptides on malignant cells. In this study, we used a mass spectrometry-based approach to identify naturally presented, CML-associated peptides in primary CML samples (HLA class I, n=21, 11,945 peptides, 5,478 source proteins; class II, n=20, 5,991 peptides, 1,302 proteins). Comparative HLA peptidome profiling using a comprehensive dataset of various benign tissues (e.g. blood, bone marrow, spleen, and lung) revealed frequently presented and strictly CML-associated antigens. In detail, the benign tissue dataset comprises hematological benign samples (class I, n=108, 51,233 peptides, 11,437 proteins; class II, n=88, 42,753 peptides, 4,877 proteins) and non-hematological benign tissues (28 tissues, n=166; class I, 128,590 peptides, 16,405 proteins; class II, 143,652 peptides, 13,410 proteins). We identified 50 CML-associated, HLA class I-restricted peptides with HLA allotype adjusted representation frequencies of ≥38% presented on HLA-A*02, -A*03, -A*11, and -B*07. HLA class II comparative profiling delineated 36 peptides exclusively and frequently presented in the HLA peptidome of ≥20% analyzed CML patients. For immunological characterization, we selected 8 HLA class I- and 6 class II-restricted highly CML-associated antigens. These peptides were analyzed in IFNγ ELISPOT assays using PBMCs from CMLTKI patients and healthy volunteers (HVs). Peptide-specific immune recognition was detected for 1/8 (13%) HLA class I peptides in 2/17 (12%) of CMLTKI patients. We hypothesized that this weak immune response might be due to an impaired CD8+ T cell function that reportedly is caused by TKI treatment. Thus, we compared T-cell responses against viral epitopes in IFNγ ELISPOT assays of CMLTKI patients with that of HVs and chronic lymphocytic leukemia (CLL) patients: in line with our hypothesis, we observed significantly reduced IFNγ release of T cells from CMLTKI patients compared to HVs and CLL patients (p<0.001), whereas CD8+ T-cell counts were not reduced. In contrast, no reduced IFNγ production was observed for HLA class II-restricted viral epitopes. These results were confirmed by memory T-cell responses detected for 6/6 (100%) HLA class II CML-associated peptides with frequencies up to 24% (4/17) of analyzed CMLTKI patients. To assess the immunogenicity of all HLA class I peptides, we performed in vitro artificial antigen presenting cell-based priming experiments using CD8+ T cells of HVs and CML patients. Effective priming of T cells was observed for 8/8 CML-associated peptides in ≥70% of analyzed HVs with frequencies of 0.1-33.9% (mean 2.2%) of CD8+ peptide-specific T cells. Notably, peptide-specific CD8+ T cells with frequencies of 0.1-2.2% (mean 0.4%) could also be induced in samples of CMLTKI patients that had not displayed preexisting immune responses. For 6/8 peptides, we observed multifunctionality of peptide-specific T cells by IFNγ and TNF production as well as upregulation of the degranulation marker CD107a. Cytotoxicity assays with polyclonal peptide-specific effector T cells confirmed the capacity to induce antigen-specific lysis for 3/4 analyzed peptides. Taken together, we here identified novel, naturally presented, CML-associated antigens and validated them as promising targets for tailored T cell-based immunotherapeutic approaches for CML patients. Disclosures Salih: Several patent applications: Patents & Royalties: e.g. EP3064507A1. Kowalewski:Immatics Biotechnologies GmbH: Employment. Schuster:Immatics Biotechnologies GmbH: Employment. Brümmendorf:Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy. Niederwieser:Miltenyi: Speakers Bureau; Novartis: Research Funding.

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.

Sign in / Sign up

Export Citation Format

Share Document