It is generally assumed that human differentiated cells have a limited life-span and proliferation capacity in vivo, and that genetic modifications are a prerequisite for their immortalization in vitro. Here we readdress this issue, studying the long-term proliferation potential of human B cells. It was shown earlier that human B cells from peripheral blood of healthy donors can be efficiently induced to proliferate for up to ten weeks in vitro by stimulating their receptor CD40 in the presence of interleukin-4. When we applied the same stimuli under conditions of modified cell number and culture size, we were surprised to find that our treatment induced B cells to proliferate throughout an observation period of presently up to 1650 days, representing more than 370 population doublings, which suggested that these B cells were immortalized in vitro. Long-term CD40-stimulated B cell cultures could be established from most healthy adult human donors. These B cells had a constant phenotype, were free from Epstein-Barr virus, and remained dependent on CD40 ligation. They had constitutive telomerase activity and stabilized telomere length. Moreover, they were susceptible to activation by Toll-like receptor 9 ligands, and could be used to expand antigen-specific cytotoxic T cells in vitro. Our results indicate that human somatic cells can evade senescence and be conditionally immortalized by external stimulation only, without a requirement for genetic manipulation or oncoviral infection. Conditionally immortalized human B cells are a new tool for immunotherapy and studies of B cell oncogenesis, activation, and function.

Major histocompatibility complex (MHC) class II alleles HLA-DQ8 and the mouse homologue I-A g7 lacking a canonical aspartic acid residue at position 57 are associated with coeliac disease 1,2 and type I diabetes 3,4 . However, the role of this single polymorphism in disease initiation and progression remains poorly understood. The lack of Asp 57 creates a positively charged P9 pocket, which confers a preference for negatively charged peptides. Gluten lacks such peptides, but tissue transglutaminase (TG2) introduces negatively charged residues at defined positions into gluten T-cell epitopes by deamidating specific glutamine residues 5,6 on the basis of their spacing to proline residues 7 . The commonly accepted model, proposing that HLA-DQ8 simply favours binding of negatively charged peptides, does not take into account the fact that TG2 requires inflammation for activation 8 and that T-cell responses against native gluten peptides are found 9,10 , particularly in children 11 . Here we show that 57 polymorphism promotes the recruitment of Tcell receptors bearing a negative signature charge in the complementary determining region 3 (CDR3 ) during the response against native gluten peptides presented by HLA-DQ8 in coeliac disease. These T cells showed a crossreactive and heteroclitic (stronger) response to deamidated gluten peptides. Furthermore, gluten peptide deamidation extended the T-cell-receptor repertoire by relieving the requirement for a charged residue in CDR3 . Thus, the lack of a negative charge at position 57 in MHC class II was met by negatively charged residues in the T-cell receptor or in the peptide, the combination of which might explain the role of HLA-DQ8 in amplifying the Tcell response against dietary gluten. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptA specific assessment of the role of MHC class II alleles in antigen-specific T-cell responses is possible in coeliac disease, because it is caused by a single and well-defined antigen, gluten, which is present in wheat, rye and barley (reviewed in ref. 12). Gluten is rich in glutamine and proline, and the high content of proline renders gluten resistant to intestinal enzymatic digestion, explaining the persistence of intact peptides for immune recognition. Notably, despite the presence of few negatively charged amino acid residues in gluten and the expression of other MHC alleles potentially capable of presenting gluten peptides, all gluten-specific CD4 + T cells isolated from the intestinal mucosa of adult patients are restricted by HLA-DQ2 or HLA-DQ8 (refs 10, 13 and 14), and most recognize a dominant peptide with a deamidated Gln-that is, a negatively charged Glu residue 5,6 .To study the T-cell response to native and deamidated gluten peptides (Fig. 1a), we immunized humanized HLA-DQ8 mice with the native 24-amino-acid gluten peptide 219-242 from the 2 (AJ133612) gliadin comprising the commonly recognized DQ8--I epitope (QGSFQPSQQ) 10 , or with a peptide deamidated at positions 229 and 237, the known ...

Celiac disease is an enteropathy caused by intolerance to dietary gluten. The disorder is strongly associated with DQA1*0501/DQB1*0201 (HLA-DQ2) as ∼95% of celiac patients express this molecule. HLA-DQ2 has unique Ag-binding properties that allow it to present a diverse set of gluten peptides to gluten-reactive CD4+ T cells so instigating an inflammatory reaction. Previous work has indicated that the presence of negatively charged amino acids within gluten peptides is required for specific binding. This, however, only partly explains the scale of the interaction. We have now characterized 432 natural ligands of HLA-DQ2 representing length variants of 155 distinct sequences. The sequences were aligned and the binding cores were inferred. Analysis of the amino acid distribution of these cores demonstrated that negatively charged residues in HLA-DQ2-bound peptides are favored at virtually all positions. This contrasts with a more restricted presence of such amino acids in T cell epitopes from gluten. Yet, HLA-DQ2 was also found to display a strong preference for proline at several anchor and nonanchor positions that largely match the position of proline in gluten T cell epitopes. Consequently, the bias for proline at p6 and p8 facilitates the enzymatic conversion of glutamine into glutamic acid in gluten peptides at p4 and p6, two important anchor sites. These observations provide new insights in the unique ability of HLA-DQ2 to bind a large repertoire of glutamine- and proline-rich gluten peptides. This knowledge may be an important asset in the development of future treatment strategies.

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Efficient protocols to generate cytomegalovirus (CMV)-specific T cells are required for adoptive immunotherapy. Recombinant Epstein-Barr virus (EBV) vectors called mini-EBV can be used to establish permanent B cell lines in a single step, which present the CMV antigen pp65 in a constitutive manner. These B cell lines, coined pp65 mini-LCL, were successfully used to reactivate and expand CMV-specific cytotoxic T cells. Here we evaluate this pp65 mini-EBV system in closer detail, focusing on (1) the quantification of T cells with specific effector function and (2) the identification of CMV-specific CD4 + helper T cells. The co-expansion of various functional CMV epitope specificities was demonstrated by IFN-c enzyme-linked immunospot assay (ELISPOT) assays and HLApeptide tetramer staining. Single-cell cloning resulted in both CD4 + and CD8 + T cell clones, the majority of which was CMV specific. Thus, mini-LCL present the pp65 antigen on HLA class I and II, mobilizing both arms of the T cell response. Using a peptide library covering the pp65 sequence for further analysis of T cell clones, we identified new pp65 CD8 + and CD4 + T cell epitopes.

During assembly, HLA class II molecules associate with the invariant chain. As the result, the peptide-binding groove is occupied by an invariant chain peptide termed CLIP (class-II-associated invariant chain peptide; sequence MRMATPLLM). By mass spectrometry, we have now characterized peptides that are naturally present in HLA-DQ2. This analysis revealed that 22 variants of Ii-derived peptides are associated with HLA-DQ2. Strikingly, the large majority of those do not contain the conventional CLIP sequence MRMATPLLM, but instead a peptide that partially overlaps with CLIP, sequence TPLLMQALPM. Peptide binding studies indicate that this alternative CLIP peptide has superior HLA-DQ2 binding properties compared to the conventional CLIP and that the minimal nine-amino-acid binding core consists of the sequence PLLMQALPM, findings that could be corroborated by molecular simulation. The alternative CLIP peptide was also found to be present in HLA-DQ2 molecules isolated from human thymus. Moreover, the alternative CLIP peptide was also found in association with HLA-DQ8. Together, these results indicate that HLA-DQ2 and HLA-DQ8 associate with an alternative CLIP sequence, a property that may relate to the strong association between HLA-DQ molecules and human autoimmune diseases.

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