Identification and Affinity Determination of Protein-Antibody and Protein-Aptamer Epitopes by Biosensor-Mass Spectrometry Combination

Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (K_D) of a protein–antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled (“conformational”) antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA–aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.     

Mapping and Characterization of HCMV-Specific Unconventional HLA-E-Restricted CD8 T Cell Populations and Associated NK and T Cell Responses Using HLA/Peptide Tetramers and Spectral Flow Cytometry

HCMV drives complex and multiple cellular immune responses, which causes a persistent immune imprint in hosts. This study aimed to achieve both a quantitative determination of the frequency for various anti-HCMV immune cell subsets, including CD8 T, γδT, NK cells, and a qualitative analysis of their phenotype. To map the various anti-HCMV cellular responses, we used a combination of three HLA_peptide tetramer complexes (HLA-E_VMAPRTLIL, HLA-E_VMAPRSLLL, and HLA-A2_NLVPMVATV) and antibodies for 18 surface markers (CD3, CD4, CD8, CD16, CD19, CD45RA, CD56, CD57, CD158, NKG2A, NKG2C, CCR7, TCRγδ, TCRγδ2, CX3CR1, KLRG1, 2B4, and PD-1) in a 20-color spectral flow cytometry analysis. This immunostaining protocol was applied to PBMCs isolated from HCMV⁻ and HCMV⁺ individuals. Our workflow allows the efficient determination of events featuring HCMV infection such as CD4/CD8 ratio, CD8 inflation and differentiation, HCMV peptide-specific HLA-E_UL40 and HLA-A2_pp65CD8 T cells, and expansion of γδT and NK subsets including δ2⁻γT and memory-like NKG2C⁺CD57⁺ NK cells. Each subset can be further characterized by the expression of 2B4, PD-1, KLRG1, CD45RA, CCR7, CD158, and NKG2A to achieve a fine-tuned mapping of HCMV immune responses. This assay should be useful for the analysis and monitoring of T-and NK cell responses to HCMV infection or vaccines.     

T Cells in Multisystem Inflammatory Syndrome in Children (MIS-C) Have a Predominant CD4+ T Helper Response to SARS-CoV-2 Peptides and Numerous Virus-Specific CD4− CD8− Double-Negative T Cells

We studied SARS-CoV-2-specific T cell responses in 22 subacute MIS-C children enrolled in 2021 and 2022 using peptide pools derived from SARS-CoV-2 spike or nonspike proteins. CD4+ and CD8+ SARS-CoV-2-specific T cells were detected in 5 subjects, CD4+ T helper (Th) responses alone were detected in 12 subjects, and CD8+ cytotoxic T cell (CTL) responses alone were documented in 1 subject. Notably, a sizeable subpopulation of CD4− CD8− double-negative (DN) T cells out of total CD3+ T cells was observed in MIS-C (median: 14.5%; IQR 8.65–25.3) and recognized SARS-CoV-2 peptides. T cells bearing the Vβ21.3 T cell receptor (TcRs), previously reported as pathogenic in the context of MIS-C, were detected in high frequencies, namely, in 2.8% and 3.9% of the CD4+ and CD8+ T cells, respectively. However, Vβ21.3 CD8+ T cells that responded to SARS-CoV-2 peptides were detected in only a single subject, suggesting recognition of nonviral antigens in the majority of subjects. Subjects studied 6–14 months after MIS-C showed T cell epitope spreading, meaning the activation of T cells that recognize more SARS-CoV-2 peptides following the initial expansion of T cells that see immunodominant epitopes. For example, subjects that did not recognize nonspike proteins in the subacute phase of MIS-C showed good Th response to nonspike peptides, and/or CD8+ T cell responses not appreciable before arose over time and could be detected in the 6–14 months’ follow-up. The magnitude of the Th and CTL responses also increased over time. In summary, patients with MIS-C associated with acute lymphopenia, a classical feature of MIS-C, showed a physiological response to the virus with a prominent role for virus-specific DN T cells.     

The Positive and Negative Immunoregulatory Role of B7 Family: Promising Novel Targets in Gastric Cancer Treatment

Gastric cancer (GC), with a heterogeneous nature, is the third leading cause of death worldwide. Over the past few decades, stable reductions in the incidence of GC have been observed. However, due to the poor response to common treatments and late diagnosis, this cancer is still considered one of the lethal cancers. Emerging methods such as immunotherapy with immune checkpoint inhibitors (ICIs) have transformed the landscape of treatment for GC patients. There are presently eleven known members of the B7 family as immune checkpoint molecules: B7-1 (CD80), B7-2 (CD86), B7-H1 (PD-L1, CD274), B7-DC (PDCD1LG2, PD-L2, CD273), B7-H2 (B7RP1, ICOS-L, CD275), B7-H3 (CD276), B7-H4 (B7x, B7S1, Vtcn1), B7-H5 (VISTA, Gi24, DD1α, Dies1 SISP1), B7-H6 (NCR3LG1), B7-H7 (HHLA2), and Ig-like domain-containing receptor 2 (ILDR2). Interaction of the B7 family of immune-regulatory ligands with the corresponding receptors resulted in the induction and inhibition of T cell responses by sending co-stimulatory and co-inhibitory signals, respectively. Manipulation of the signals provided by the B7 family has significant potential in the management of GC.     

Complementary Effects of Carbamylated and Citrullinated LL37 in Autoimmunity and Inflammation in Systemic Lupus Erythematosus

LL37 acts as T-cell/B-cell autoantigen in Systemic lupus erythematosus (SLE) and psoriatic disease. Moreover, when bound to “self” nucleic acids, LL37 acts as “danger signal,” leading to type I interferon (IFN-I)/pro-inflammatory factors production. T-cell epitopes derived from citrullinated-LL37 act as better antigens than unmodified LL37 epitopes in SLE, at least in selected HLA-backgrounds, included the SLE-associated HLA-DRB1*1501/HLA-DRB5*0101 backgrounds. Remarkably, while “fully-citrullinated” LL37 acts as better T-cell-stimulator, it loses DNA-binding ability and the associated “adjuvant-like” properties. Since LL37 undergoes a further irreversible post-translational modification, carbamylation and antibodies to carbamylated self-proteins other than LL37 are present in SLE, here we addressed the involvement of carbamylated-LL37 in autoimmunity and inflammation in SLE. We detected carbamylated-LL37 in SLE-affected tissues. Most importantly, carbamylated-LL37-specific antibodies and CD4 T-cells circulate in SLE and both correlate with disease activity. In contrast to “fully citrullinated-LL37,” “fully carbamylated-LL37” maintains both innate and adaptive immune-cells’ stimulatory abilities: in complex with DNA, carbamylated-LL37 stimulates plasmacytoid dendritic cell IFN-α production and B-cell maturation into plasma cells. Thus, we report a further example of how different post-translational modifications of a self-antigen exert complementary effects that sustain autoimmunity and inflammation, respectively. These data also show that T/B-cell responses to carbamylated-LL37 represent novel SLE disease biomarkers.     

Interactions between immunoglobulin-like and catalytic modules in Clostridium thermocellum cellulosomal cellobiohydrolase CbhA

Cellobiohydrolase CbhA from Clostridium thermocellum cellulosome is a multi-modular protein composed starting from the N-terminus of a carbohydrate-binding module (CBM) of family 4, an immunoglobulin(Ig)-like module, a catalytic module of family 9 glycoside hydrolases (GH9), X1(1) and X1(2) modules, a CBM of family 3 and a dockerin module. Deletion of the Ig-like module from the Ig-GH9 construct results in complete inactivation of the GH9 module. The crystal structure of the Ig-GH9 module pair reveals the existence of an extensive module interface composed of over 40 amino acid residues of both modules and maintained through a large number of hydrophilic and hydrophobic interactions. To investigate the importance of these interactions between the two modules, we compared the secondary and tertiary structures and thermostabilities of the individual Ig-like and GH9 modules and the Ig-GH9 module pair using both circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). Thr230, Asp262 and Asp264 of the Ig-like module are located in the module interface of the Ig-GH9 module pair and are suggested to be important in 'communication' between the modules. These residues were mutated to alanyl residues. The structure, stability and catalytic properties of the native Ig-GH9 and its D264A and T230A/D262A mutants were compared. The results indicate that despite being able to fold relatively independently, the Ig-like and GH9 modules interact and these interactions affect the final fold and stability of each module. Mutations of one or two amino acid residues lead to destabilization and change of the mechanism of thermal unfolding of the polypeptides. The enzymatic properties of native Ig-GH9, D264A and T230A/D262A mutants are similar. The results indicate that inactivation of the GH9 module occurs as a result of multiple structural disturbances finally affecting the topology of the catalytic center.     

Ceramide Synthase 2 Null Mice Are Protected from Ovalbumin-Induced Asthma with Higher T Cell Receptor Signal Strength in CD4+ T Cells

(1) Background: six mammalian ceramide synthases (CerS1–6) determine the acyl chain length of sphingolipids (SLs). Although ceramide levels are increased in murine allergic asthma models and in asthmatic patients, the precise role of SLs with specific chain lengths is still unclear. The role of CerS2, which mainly synthesizes C22–C24 ceramides, was investigated in immune responses elicited by airway inflammation using CerS2 null mice. (2) Methods: asthma was induced in wild type (WT) and CerS2 null mice with ovalbumin (OVA), and inflammatory cytokines and CD4 (cluster of differentiation 4)+ T helper (Th) cell profiles were analyzed. We also compared the functional capacity of CD4+ T cells isolated from WT and CerS2 null mice. (3) Results: CerS2 null mice exhibited milder symptoms and lower Th2 responses than WT mice after OVA exposure. CerS2 null CD4+ T cells showed impaired Th2 and increased Th17 responses with concomitant higher T cell receptor (TCR) signal strength after TCR stimulation. Notably, increased Th17 responses of CerS2 null CD4+ T cells appeared only in TCR-mediated, but not in TCR-independent, treatment. (4) Conclusions: altered Th2/Th17 immune response with higher TCR signal strength was observed in CerS2 null CD4+ T cells upon TCR stimulation. CerS2 and very-long chain SLs may be therapeutic targets for Th2-related diseases such as asthma.     

Human CD4+CD45RA+ T Cells Behavior after In Vitro Activation: Modulatory Role of Vasoactive Intestinal Peptide

Naїve CD4⁺ T cells, which suffer different polarizing signals during T cell receptor activation, are responsible for an adequate immune response. In this study, we aimed to evaluate the behavior of human CD4⁺CD45RA⁺ T cells after in vitro activation by anti-CD3/CD28 bead stimulation for 14 days. We also wanted to check the role of the VIP system during this process. The metabolic biomarker Glut1 was increased, pointing to an increase in glucose requirement whereas Hif-1α expression was higher in resting than in activated cells. Expression of Th1 markers increased at the beginning of activation, whereas Th17-associated biomarkers augmented after that, showing a pathogenic Th17 profile with a possible plasticity to Th17/1. Foxp3 mRNA expression augmented from day 4, but no parallel increases were observed in IL-10, IL-2, or TGFβ mRNA expression, meaning that these potential differentiated Treg could not be functional. Both VIP receptors were located on the plasma membrane, and expression of VPAC₂ receptor increased significantly with respect to the VPAC₁ receptor from day 4 of CD4⁺CD45RA⁺ T activation, pointing to a shift in VPAC receptors. VIP decreased IFNγ and IL-23R expression during the activation, suggesting a feasible modulation of Th17/1 plasticity and Th17 stabilization through both VPAC receptors. These novel results show that, without polarizing conditions, CD4⁺CD45RA⁺ T cells differentiate mainly to a pathogenic Th17 subset and an unpaired Treg subset after several days of activation. Moreover, they confirm the important immunomodulatory role of VIP, also on naїve Th cells, stressing the importance of this neuropeptide on lymphocyte responses in different pathological or non-pathological situations.     

Reduced Expression of PD-1 in Circulating CD4+ and CD8+ Tregs Is an Early Feature of RRMS

Altered regulatory T cell (Treg) function could contribute to MS. The expression of activating and inhibitory receptors influences the activity of Tregs. Our aim was to investigate T cell phenotypes in relapsing–remitting MS (RRMS) patients at an early phase of the disease. We examined the influence of demographic parameters on the distribution of CD4+ and CD8+ T cell subclasses by generalized linear modeling. We also studied the expression of the following markers—CTLA-4, GITR, PD-1, FoxP3, Helios, CD28, CD62L, CD103—on T cell subsets from peripheral blood with a 14-color flow cytometry panel. We used an antibody array to define the profiles of 34 Th1/Th2/Th17 cytokines in the serum. Expression of PD-1 and GITR on CD4+ and CD8+ Tregs was decreased in RRMS patients. The proinflammatory factors IFN-γ, IL-17, IL-17F, TGFβ-1, TGFβ-3, IL-1SRII, IL-12 p40, sgp130, IL-6sR were significantly increased in RRMS patients. Therefore, a deficiency of PD-1 and GITR immune checkpoints on CD4+ and CD8+ Tregs is a feature of RRMS and might underlie impaired T cell control.     

A MLR-Based Approach to Analyze Regulators of T Lymphocyte Activation In Vivo

Depending on the context, robust and durable T lymphocyte activation is either desirable, as in the case of anti-tumor responses, or unwanted, in cases of autoimmunity when chronic stimulation leads to self-tissue damage. Therefore, reliable in vivo models are of great importance to identify and validate regulatory pathways of T lymphocyte activation. Here, we describe an in vivo mixed-lymphocyte-reaction (MLR) approach, which is based on the so-called parent-into-F1 (P → F1) mouse model in combination with the congenic marker CD45.1/2 and cell proliferation dye-labeling. This setup allows us to track adoptively transferred allogenic CD4⁺ and CD8⁺ T lymphocytes and analyze their phenotype as well as the proliferation by flow cytometry in the blood and spleen. We could show hypo-reactive responses of T lymphocytes isolated from knockout mice with a known defect in T lymphocyte activation. Thus, this MLR-based in vivo model provides the opportunity to analyze positive regulators of T cell responses under physiological conditions of polyclonal T lymphocyte activation in vivo.     

Cord Blood T Cells Expressing High and Low PKCζ Levels Develop into Cells with a Propensity to Display Th1 and Th9 Cytokine Profiles,Respectively

Low Protein Kinase C zeta (PKCζ) levels in cord blood T cells (CBTC) have been shown to correlate with the development of allergic sensitization in childhood. However, little is known about the mechanisms responsible. We have examined the relationship between the expression of different levels of PKCζ in CBTC and their development into mature T cell cytokine producers that relate to allergy or anti-allergy promoting cells. Maturation of naïve CBTC was initiated with anti-CD3/-CD28 antibodies and recombinant human interleukin-2 (rhIL-2). To stimulate lymphocyte proliferation and cytokine production the cells were treated with Phytohaemagglutinin (PHA) and Phorbol myristate acetate (PMA). Irrespective of the PKCζ levels expressed, immature CBTC showed no difference in lymphocyte proliferation and the production of T helper 2 (Th2) cytokine interleukin-4 (IL-4) and Th1 cytokine, interferon-gamma (IFN-γ), and influenced neither their maturation from CD45RA⁺ to CD45RO⁺ cells nor cell viability/apoptosis. However, upon maturation the low PKCζ expressing cells produced low levels of the Th1 cytokines, IFN-γ, IL-2 and tumour necrosis factor-alpha (TNF), no changes to levels of the Th2 cytokines, IL-4, IL-5 and IL-13, and an increase in the Th9 cytokine, IL-9. Other cytokines, lymphotoxin-α (LT-α), IL-10, IL-17, IL-21, IL-22 and Transforming growth factor-beta (TGF-β) were not significantly different. The findings support the view that low CBTC PKCζ levels relate to the increased risk of developing allergic diseases.     

CD40/APC-specific antibodies with three T-cell epitopes loaded in the constant domains induce CD4+ T-cell responses

CD4+ T lymphocytes play a central role in the orchestration and maintenance of the adaptive immune response. Targeting of antigen to antigen presenting cells (APCs) increases peptide loading of major histocompatibility complex (MHC) class II molecules and CD4+ T-cell activation. APCs have been targeted by APC-specific recombinant antibodies (rAbs) with single T-cell epitopes integrated in the constant region of the heavy chain (C(H)). However, the strategy may be improved if several T-cell epitopes could be delivered simultaneously by one rAb. We here demonstrate that a single rAb can be loaded with multiple identical or different T-cell epitopes, integrated as loops between β-strands in C(H) domains. One epitope was inserted in C(H)1, while two were placed in C(H)2 of IgG. T-cell proliferation assays showed that all three peptides were excised from loops and presented on MHC class II to T-cells. Induction of T-cell activation by each epitope in the multi-peptide rAb was as good, or even better, than that elicited by corresponding single-peptide rAbs. Furthermore, following DNA vaccination of mice with plasmids that encode CD40-specific rAbs loaded with either one or three peptides, T-cell responses were induced. Thus, integration of multiple epitopes in C(H) region loops of APC-specific rAbs is feasible and may be utilized in design of multi-vaccines.     

Function of IRAG2 Is Modulated by NO/cGMP in Murine Platelets

Inositol 1,4,5-triphosphate receptor-associated 2 (IRAG2) is a type II membrane protein located at the endoplasmic reticulum. It is a homologue of inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate 1 (IRAG1), a substrate protein of cGMP-dependent protein kinase I (PKGI), and is among others expressed in platelets. Here, we studied if IRAG2 is also located in platelets and might be a substrate protein of PKGI. IRAG2 was detected in platelets of IRAG2-WT animals but not in those of IRAG2-KO animals. Next, we validated by co-immunoprecipitation studies that IRAG2 is associated with IP₃R1-3. No direct stable interaction with PKGIβ or with IRAG1 was observed. Phosphorylation of IRAG2 in murine platelets using a Ser/Thr-specific phospho-antibody was found in vitro and ex vivo upon cGMP stimulation. To gain insight into the function of IRAG2, platelet aggregation studies were performed using thrombin and collagen as agonists for treatment of isolated IRAG2-WT or IRAG2-KO platelets. Interestingly, platelet aggregation was reduced in the absence of IRAG2. Pretreatment of wild type or IRAG2-KO platelets with sodium nitroprusside (SNP) or 8-pCPT-cGMP revealed a further reduction in platelet aggregation in the absence of IRAG2. These results show that IRAG2 is a substrate of PKGI in murine platelets. Furthermore, our results indicate that IRAG2 is involved in the induction of thrombin- or collagen-induced platelet aggregation and that this effect is enhanced by cGMP-dependent phosphorylation of IRAG2. As IRAG1 was previously shown to inhibit platelet aggregation in a cGMP-dependent manner, it can be speculated that IRAG2 exerts an opposing function and might be an IRAG1 counterpart in murine platelets.     

Erythroid Differentiation Regulator 1 Strengthens TCR Signaling by Enhancing PLCγ1 Signal Transduction Pathway

Erythroid differentiation regulator 1 (Erdr1) has previously been reported to control thymocyte selection via TCR signal regulation, but the effect of Erdr1 as a TCR signaling modulator was not studied in peripheral T cells. In this report, it was determined whether Erdr1 affected TCR signaling strength in CD4 T cells. Results revealed that Erdr1 significantly enhanced the anti-TCR antibody-mediated activation and proliferation of T cells while failing to activate T cells in the absence of TCR stimulation. In addition, Erdr1 amplified Ca²⁺ influx and the phosphorylation of PLCγ1 in CD4 T cells with the TCR stimuli. Furthermore, NFAT1 translocation into nuclei in CD4 T cells was also significantly promoted by Erdr1 in the presence of TCR stimulation. Taken together, our results indicate that Erdr1 positively modulates TCR signaling strength via enhancing the PLCγ1/Ca²⁺/NFAT1 signal transduction pathway.     

Improved design of an antigen with enhanced specificity for the broadly HIV-neutralizing antibody b12

In an attempt to design immunogens that elicit broadly HIV-neutralizing antibodies, we recently engineered monomeric HIV-1 gp120 to bind preferentially b12, a broadly neutralizing antibody to the CD4-binding site (CD4bs) on gp120, by mutating four central residues in the CD4bs to alanine and introducing extra N-glycosylation sites potentially to mask unwanted B-cell epitopes. Despite the favorable antigenicity of this mutant, it harbors two potential caveats that may limit its effectiveness to elicit b12-like antibodies: (i) b12-binding affinity is reduced relative to wild-type gp120 and (ii) binding of some non-neutralizing antibodies to the N-terminal C1 region of gp120 is still observed. Here, we sought to correct these potential limitations. By reverting one of the added N-glycosylation sites on the gp120 core, b12 binding was improved without affecting the epitope-masking properties of the original mutant. Furthermore, truncation of the gp120 N-terminus eliminated binding of the anti-C1 antibodies. Finally, based on the binding profiles of additional non-neutralizing antibodies tested here, further N-glycosylation sites were incorporated to mask their corresponding epitopes. The resulting hyperglycosylated gp120 variants bind b12 and another broadly neutralizing antibody, 2G12, with apparent affinities approaching that of wild-type gp120, but do not bind 21 non- or weakly neutralizing antibodies to seven different epitopes on gp120. These hyperglycosylated variants expand our panel of glycoengineered gp120s that are currently being evaluated for their ability to elicit broadly neutralizing antibodies.     

Escherichia coli Maltose-Binding Protein Induces M1 Polarity of RAW264.7 Macrophage Cells via a TLR2- and TLR4-Dependent Manner

Maltose-binding protein (MBP) is a critical player of the maltose/maltodextrin transport system in Escherichia coli. Our previous studies have revealed that MBP nonspecifically induces T helper type 1 (Th1) cell activation and activates peritoneal macrophages obtained from mouse. In the present study, we reported a direct stimulatory effect of MBP on RAW264.7 cells, a murine macrophage cell line. When stimulated with MBP, the production of nitric oxide (NO), IL-1β, IL-6 and IL-12p70, and the expressions of CD80, MHC class II and inducible nitric oxide synthase (iNOS) were all increased in RAW264.7 cells, indicating the activation and polarization of RAW264.7 cells into M1 macrophages induced by MBP. Further study showed that MBP stimulation upregulated the expression of TLR2 and TLR4 on RAW264.7 cells, which was accompanied by subsequent phosphorylation of IκB-α and p38 MAPK. Pretreatment with anti-TLR2 or anti-TLR4 antibodies largely inhibited the phosphorylation of IκB-α and p38 MAPK, and greatly reduced MBP-induced NO and IL-12p70 production, suggesting that the MBP-induced macrophage activation and polarization were mediated by TLR2 and TLR4 signaling pathways. The observed results were independent of lipopolysaccharide contamination. Our study provides a new insight into a mechanism by which MBP enhances immune responses and warrants the potential application of MBP as an immune adjuvant in immune therapies.