Cell Surface B2m-Free Human Leukocyte Antigen (HLA) Monomers and Dimers: Are They Neo-HLA Class and Proto-HLA?

Cell surface HLA-I molecules (Face-1) consist of a polypeptide heavy chain (HC) with two groove domains (G domain) and one constant domain (C-domain) as well as a light chain, B2-microglobulin (B2m). However, HCs can also independently emerge unfolded on the cell surface without peptides as B2m-free HC monomers (Face-2), B2m-free HC homodimers (Face 3), and B2m-free HC heterodimers (Face-4). The transport of these HLA variants from ER to the cell surface was confirmed by antiviral antibiotics that arrest the release of newly synthesized proteins from the ER. Face-2 occurs at low levels on the normal cell surface of the lung, bronchi, epidermis, esophagus, breast, stomach, ilium, colorectum, gall bladder, urinary bladder, seminal vesicles ovarian epithelia, endometrium, thymus, spleen, and lymphocytes. They are upregulated on immune cells upon activation by proinflammatory cytokines, anti-CD3 antibodies, antibiotics (e.g., ionomycin), phytohemagglutinin, retinoic acid, and phorbol myristate acetate. Their density on the cell surface remains high as long as the cells remain in an activated state. After activation-induced upregulation, the Face-2 molecules undergo homo- and hetero-dimerization (Face-3 and Face-4). Alterations in the redox environment promote dimerization. Heterodimerization can occur among and between the alleles of different haplotypes. The glycosylation of these variants differ from that of Face-1, and they may occur with bound exogenous peptides. Spontaneous arthritis occurs in HLA-B27+ mice lacking B2m (HLA-B27+ B2m-/-) but not in HLA-B27+ B2m+/- mice. The mice with HLA-B27 in Face-2 spontaneous configuration develop symptoms such as changes in nails and joints, hair loss, and swelling in paws, leading to ankyloses. Anti-HC-specific mAbs delay disease development. Some HLA-I polyreactive mAbs (MEM series) used for immunostaining confirm the existence of B2m-free variants in several cancer cells. The upregulation of Face-2 in human cancers occurs concomitantly with the downregulation of intact HLAs (Face-1). The HLA monomeric and dimeric variants interact with inhibitory and activating ligands (e.g., KIR), growth factors, cytokines, and neurotransmitters. Similarities in the amino acid sequences of the HLA-I variants and HLA-II ß-chain suggest that Face-2 could be the progenitor of both HLA classes. These findings may support the recognition of these variants as a neo-HLA class and proto-HLA.

Antibodies for ß2-Microglobulin and the Heavy Chains of HLA-E, HLA-F, and HLA-G Reflect the HLA-Variants on Activated Immune Cells and Phases of Disease Progression in Rheumatoid Arthritis Patients under Treatment.

Rheumatoid arthritis (RA) is a progressive, inflammatory, autoimmune, symmetrical polyarticular arthritis. It is characterized by synovial infiltration and activation of several types of immune cells, culminating in their apoptosis and antibody generation against "altered" autoantigens. ß2-microglobulin (ß2m)-associated heavy chains (HCs) of HLA antigens, also known as closed conformers (Face-1), undergo "alteration" during activation of immune cells, resulting in ß2m-free structural variants, including monomeric open conformers (Face-2) that are capable of dimerizing as either homodimers (Face-3) or as heterodimers (Face-4). ß2m-free HCs uncover the cryptic epitopes that can elicit antibodies (Abs). We report here the levels of IgM and IgG Abs against both ß2m and HCs of HLA-E, HLA-F, and HLA-G in 74 RA patients receiving immunosuppressive drugs. Anti-ß2m IgM was present in 20 of 74 patients, whereas anti-ß2m IgG was found in only 8 patients. Abs against ß2m would be expected if Abs were generated against ß2m-associated HLA HCs. The majority of patients were devoid of either anti-ß2m IgM or IgG but had Abs against HCs of different HLA-Ib molecules. The paucity of anti-ß2m Abs in this cohort of patients suggests that Abs were developed against ß2m-free HLA HCs, such as Face-2, Face-3, and Face-4. While 63 of 68 patients had IgG Abs against anti-HLA-F HCs, 36 and 50 patients showed IgG Ab reactivity against HLA-E and anti-HLA-G HCs, respectively. Evidently, anti-HLA-F HC Abs are the most predominant anti-HLA-Ib HC IgG Abs in RA patients. The incidence and intensity of Abs against HLA-E, HLA-F, and HLA-G in the normal control group were much higher than those observed in RA patients. Evidently, the lower level of Abs in RA patients points to the impact of the immunosuppressive drugs on these patients. These results underscore the need for further studies to unravel the nature of HLA-F variants on activated immune cells and synoviocytes of RA patients.

Ramifications of the HLA-I Allelic Reactivity of Anti-HLA-E*01:01 and Anti-HLA-E*01:03 Heavy Chain Monoclonal Antibodies in Comparison with Anti-HLA-I IgG Reactivity in Non-Alloimmunized Males, Melanoma-Vaccine Recipients, and End-Stage Renal Disease Patients.

Serum anti-HLA-I IgG are present in non-alloimmunized males, cancer patients, and transplant recipients. Anti-HLA-I antibodies are also present in intravenous immunoglobulin (IVIg), prepared from the plasma of thousands of healthy donors. However, the HLA-Ia reactivity of IVIg diminishes markedly after passing through HLA-E HC-affinity columns, suggesting that the HLA-I reactivity is due to antibodies formed against HLA-E. Hence, we examined whether anti-HLA-E antibodies can react to HLA-I alleles. Monoclonal IgG antibodies (mAbs) against HCs of two HLA-E alleles were generated in Balb/C mice. The antibodies were analyzed using multiplex bead assays on a Luminex platform for HLA-I reactivity. Beads coated with an array of HLA heterodimers admixed with HCs (LABScreen) were used to examine the binding of IgG to different HLA-Ia (31-HLA-A, 50-HLA-B, and 16-HLA-C) and Ib (2-HLA-E, one each of HLA-F and HLA-G) alleles. A striking diversity in the HLA-Ia and/or HLA-Ib reactivity of mAbs was observed. The number of the mAbs reactive to (1) only HLA-E (n = 25); (2) all HLA-Ib isomers (n = 8); (3) HLA-E and HLA-B (n = 5); (4) HLA-E, HLA-B, and HLA-C (n = 30); (5) HLA-E, HLA-A*1101, HLA-B, and HLA-C (n = 83); (6) HLA-E, HLA-A, HLA-B, and HLA-C (n = 54); and (7) HLA-Ib and HLA-Ia (n = 8), in addition to four other minor groups. Monospecificity and polyreactivity were corroborated by HLA-E monospecific and HLA-I shared sequences. The diverse HLA-I reactivity of the mAbs are compared with the pattern of HLA-I reactivity of serum-IgG in non-alloimmunized males, cancer patients, and ESKD patients. The findings unravel the diagnostic potential of the HLA-E monospecific-mAbs and immunomodulatory potentials of IVIg highly mimicking HLA-I polyreactive-mAbs.

Four Faces of Cell-Surface HLA Class-I: Their Antigenic and Immunogenic Divergence Generating Novel Targets for Vaccines.

Leukocyte cell-surface HLA-I molecules, involved in antigen presentation of peptides to CD8+ T-cells, consist of a heavy chain (HC) non-covalently linked to ß2-microglobulin (ß2m) (Face-1). The HC amino acid composition varies across all six isoforms of HLA-I, while that of ß2m remains the same. Each HLA-allele differs in one or more amino acid sequences on the HC α1 and α2 helices, while several sequences among the three helices are conserved. HCs without ß2m (Face-2) are also observed on human cells activated by malignancy, viral transformation, and cytokine or chemokine-mediated inflammation. In the absence of ß2m, the monomeric Face-2 exposes immunogenic cryptic sequences on these cells as confirmed by HLA-I monoclonal antibodies (LA45, L31, TFL-006, and TFL-007). Furthermore, such exposure enables dimerization between two Face-2 molecules by SH-linkage, salt linkage, H-bonding, and van der Waal forces. In HLA-B27, the linkage between two heavy chains with cysteines at position of 67 of the amino acid residues was documented. Similarly, several alleles of HLA-A, B, C, E, F and G express cysteine at 67, 101, and 164, and additionally, HLA-G expresses cysteine at position 42. Thus, the monomeric HC (Face-2) can dimerize with another HC of its own allele, as homodimers (Face-3), or with a different HC-allele, as heterodimers (Face-4). The presence of Face-4 is well documented in HLA-F. The post-translational HLA-variants devoid of ß2m may expose several cryptic linear and non-linear conformationally altered sequences to generate novel epitopes. The objective of this review, while unequivocally confirming the post-translational variants of HLA-I, is to highlight the scientific and clinical importance of the four faces of HLA and to prompt further research to elucidate their functions and their interaction with non-HLA molecules during inflammation, infection, malignancy and transplantation. Indeed, these HLA faces may constitute novel targets for passive and active specific immunotherapy and vaccines.

Role of HLA-I Structural Variants and the Polyreactive Antibodies They Generate in Immune Homeostasis.

Cell-surface HLA-I molecules consisting of ß2-microglobulin (ß2m) associated heavy chains (HCs), referred to as Face-1, primarily present peptides to CD8+ T-cells. HCs consist of three α-domains, with selected amino acid sequences shared by all alleles of all six isoforms. The cell-surface HLA undergoes changes upon activation by pathological conditions with the expression of ß2m-free HCs (Face-2) resulting in exposure of ß2m-masked sequences shared by almost all alleles and the generation of HLA-polyreactive antibodies (Abs) against them. Face-2 may homodimerize or heterodimerize with the same (Face-3) or different alleles (Face-4) preventing exposure of shared epitopes. Non-allo immunized males naturally carry HLA-polyreactive Abs. The therapeutic intravenous immunoglobulin (IVIg) purified from plasma of thousands of donors contains HLA-polyreactive Abs, admixed with non-HLA Abs. Purified HLA-polyreactive monoclonal Abs (TFL-006/007) generated in mice after immunizing with Face-2 are documented to be immunoregulatory by suppressing or activating different human lymphocytes, much better than IVIg. Our objectives are (a) to elucidate the complexity of the HLA-I structural variants, and their Abs that bind to both shared and uncommon epitopes on different variants, and (b) to examine the roles of those Abs against HLA-variants in maintaining immune homeostasis. These may enable the development of personalized therapeutic strategies for various pathological conditions.

Functional Profiling of Head and Neck/Esophageal Squamous Cell Carcinoma to Predict Cetuximab Response.

Background: Cetuximab, an epidermal growth factor receptor (EGFR)-targeting antibody, remains the only Food and Drug Administration-approved targeted therapy for squamous cell carcinoma (SCC) of head and neck/esophagus. However, in clinical trials, cetuximab only benefited a subset of patients and frequently caused toxicity. Predicting which patients respond to cetuximab remains unsolved. The authors sought to identify predictive biomarkers in EGFR signaling and autophagy pathways, which may be impacted by cetuximab under certain treatment conditions. Methods: In vitro responses of SCC cell lines to cetuximab under various nutrient conditions were assessed by WST-8 growth assay. Functional profiles of several EGFR signaling biomarkers were investigated by Luminex-based assays and corroborated with immunoblots. Autophagy markers were analyzed with immunoblots. Results: In vitro growth response assays identified cetuximab responder and nonresponder cell lines. Optimal growth conditions and growth factors enhanced responses, and even reversed nonresponsiveness in some cell lines. Strong correlation was found between response in growth assays (reference assay) and dynamic changes in p-Erk1/2 and LC3-II (index assays). Conclusions: This study indicates that nutrient modification may enhance cetuximab response in SCC patients. Biomarker results strengthen the hypothesis that dynamic biomarkers can be used to predict patient response to cetuximab. Future studies are warranted to test in more complex samples including patient-derived tumor tissues.

SITC cancer immunotherapy resource document: a compass in the land of biomarker discovery.

Since the publication of the Society for Immunotherapy of Cancer's (SITC) original cancer immunotherapy biomarkers resource document, there have been remarkable breakthroughs in cancer immunotherapy, in particular the development and approval of immune checkpoint inhibitors, engineered cellular therapies, and tumor vaccines to unleash antitumor immune activity. The most notable feature of these breakthroughs is the achievement of durable clinical responses in some patients, enabling long-term survival. These durable responses have been noted in tumor types that were not previously considered immunotherapy-sensitive, suggesting that all patients with cancer may have the potential to benefit from immunotherapy. However, a persistent challenge in the field is the fact that only a minority of patients respond to immunotherapy, especially those therapies that rely on endogenous immune activation such as checkpoint inhibitors and vaccination due to the complex and heterogeneous immune escape mechanisms which can develop in each patient. Therefore, the development of robust biomarkers for each immunotherapy strategy, enabling rational patient selection and the design of precise combination therapies, is key for the continued success and improvement of immunotherapy. In this document, we summarize and update established biomarkers, guidelines, and regulatory considerations for clinical immune biomarker development, discuss well-known and novel technologies for biomarker discovery and validation, and provide tools and resources that can be used by the biomarker research community to facilitate the continued development of immuno-oncology and aid in the goal of durable responses in all patients.

Monitoring response to treatment in melanoma patients: potential of a serum glycomic marker.

A mechanistic marker correlating with tumor progression and clinical response is useful for assessing therapeutic response and determining the course of therapy. Since serum-total-ganglioside (sTG) and antiganglioside-IgM antibody levels reflected tumor progression, the feasibility of utilizing sTG for assessing the response to immunotherapy of metastatic-melanoma was tested. Patients (n = 34) were immunized with dendritic cells cocultured with irradiated, IFN gamma-treated autologous tumor cells admixed with GM-CSF. Levels of sTG and antiganglioside-IgM antibody titers were measured in sera of vaccine recipients at 0, 4 and 24 weeks of treatment. Based on sTG-level, whether lower (L) or higher (H) than the mean + 1 SD of normal and healthy volunteers on weeks 0, 4 and 24, patients were categorized into cohorts-I (LLL, n = 16), II (HHL/HLL, n = 4), III (LLH/LHH/LHL, n = 7) and IV (HHH/HLH, n = 7). The cohorts were regrouped as sTG- downregulators (sTG-DR; n = 20) and upregulators (sTG-UR; n = 14). These two cohorts differed significantly in their overall (p < 0.012) and progression-free (p = 0.0001) survival post-treatment. 43% sTG-UR died within 39 months, with a median survival of 39 months, whereas 61% of the sTG-DR survived for 48 months. Both endogenous and vaccine-induced antiganglioside-IgM antibodies appeared to regulate sTG levels. Nonresponders had increased sTG with no or low IgM antibody response. The sTG level is regulated within 24 weeks post-treatment and therefore, may serve as an ideal biomarker for assessing therapeutic responses in patients. Clinical correlations of sTG indicate that sTG-downregulating therapy may be an effective treatment strategy for melanoma.

Proteomic profiles by multiplex microsphere suspension array.

Advances in high-throughput proteomic approaches have provided substantial momentum to novel disease-biomarker discovery research and have augmented the quality of clinical studies. Applications based on multiplexed microsphere suspension array technology are making strong in-roads into the clinical diagnostic/prognostic practice. Conventional proteomic approaches are designed to discover a broad set of proteins that are associated with a specific medical condition. In comparison, multiplex microsphere immunoassays use quantitative measurements of selected set(s) of specific/particular molecular markers such as cytokines, chemokines, pathway signaling or disease-specific markers for detection, metabolic disorders, cancer, and infectious agents causing human, plant and animal diseases. This article provides a foundation to the multiplexed microsphere suspension array technology, with an emphasis on the improvements in the technology, data analysis approaches, and applications to translational and clinical research with implications for personalized and precision medicine.

Indoleamine 2,3-dioxygenase (IDO): Biology and Target in Cancer Immunotherapies.

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing oxidoreductase that catalyzes the initial and rate-limiting step in the breakdown of non-dietary tryptophan. The biology and immunomodulatory role for IDO is discussed in this review with a focus on its interaction with immune cells and its potential therapeutic target in the clinic. IDO has been revealed to be a central regulator of immune responses in a broad variety of physiological and pathological settings, mostly serving as a multifaceted negative feedback mechanism, to self-regulate immune responses. IDO is considered a therapeutic target in cancer and the use of IDO inhibitors as single agent or in combination with other treatment modalities are under active investigation.

Validation of biomarkers to predict response to immunotherapy in cancer: Volume I - pre-analytical and analytical validation.

Immunotherapies have emerged as one of the most promising approaches to treat patients with cancer. Recently, there have been many clinical successes using checkpoint receptor blockade, including T cell inhibitory receptors such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death-1 (PD-1). Despite demonstrated successes in a variety of malignancies, responses only typically occur in a minority of patients in any given histology. Additionally, treatment is associated with inflammatory toxicity and high cost. Therefore, determining which patients would derive clinical benefit from immunotherapy is a compelling clinical question. Although numerous candidate biomarkers have been described, there are currently three FDA-approved assays based on PD-1 ligand expression (PD-L1) that have been clinically validated to identify patients who are more likely to benefit from a single-agent anti-PD-1/PD-L1 therapy. Because of the complexity of the immune response and tumor biology, it is unlikely that a single biomarker will be sufficient to predict clinical outcomes in response to immune-targeted therapy. Rather, the integration of multiple tumor and immune response parameters, such as protein expression, genomics, and transcriptomics, may be necessary for accurate prediction of clinical benefit. Before a candidate biomarker and/or new technology can be used in a clinical setting, several steps are necessary to demonstrate its clinical validity. Although regulatory guidelines provide general roadmaps for the validation process, their applicability to biomarkers in the cancer immunotherapy field is somewhat limited. Thus, Working Group 1 (WG1) of the Society for Immunotherapy of Cancer (SITC) Immune Biomarkers Task Force convened to address this need. In this two volume series, we discuss pre-analytical and analytical (Volume I) as well as clinical and regulatory (Volume II) aspects of the validation process as applied to predictive biomarkers for cancer immunotherapy. To illustrate the requirements for validation, we discuss examples of biomarker assays that have shown preliminary evidence of an association with clinical benefit from immunotherapeutic interventions. The scope includes only those assays and technologies that have established a certain level of validation for clinical use (fit-for-purpose). Recommendations to meet challenges and strategies to guide the choice of analytical and clinical validation design for specific assays are also provided.

Validation of biomarkers to predict response to immunotherapy in cancer: Volume II - clinical validation and regulatory considerations.

There is growing recognition that immunotherapy is likely to significantly improve health outcomes for cancer patients in the coming years. Currently, while a subset of patients experience substantial clinical benefit in response to different immunotherapeutic approaches, the majority of patients do not but are still exposed to the significant drug toxicities. Therefore, a growing need for the development and clinical use of predictive biomarkers exists in the field of cancer immunotherapy. Predictive cancer biomarkers can be used to identify the patients who are or who are not likely to derive benefit from specific therapeutic approaches. In order to be applicable in a clinical setting, predictive biomarkers must be carefully shepherded through a step-wise, highly regulated developmental process. Volume I of this two-volume document focused on the pre-analytical and analytical phases of the biomarker development process, by providing background, examples and "good practice" recommendations. In the current Volume II, the focus is on the clinical validation, validation of clinical utility and regulatory considerations for biomarker development. Together, this two volume series is meant to provide guidance on the entire biomarker development process, with a particular focus on the unique aspects of developing immune-based biomarkers. Specifically, knowledge about the challenges to clinical validation of predictive biomarkers, which has been gained from numerous successes and failures in other contexts, will be reviewed together with statistical methodological issues related to bias and overfitting. The different trial designs used for the clinical validation of biomarkers will also be discussed, as the selection of clinical metrics and endpoints becomes critical to establish the clinical utility of the biomarker during the clinical validation phase of the biomarker development. Finally, the regulatory aspects of submission of biomarker assays to the U.S. Food and Drug Administration as well as regulatory considerations in the European Union will be covered.

"Adherent" versus Other Isolation Strategies for Expanding Purified, Potent, and Activated Human NK Cells for Cancer Immunotherapy.

Natural killer (NK) cells have long been hypothesized to play a central role in the development of new immunotherapies to combat a variety of cancers due to their intrinsic ability to lyse tumor cells. For the past several decades, various isolation and expansion methods have been developed to harness the full antitumor potential of NK cells. These protocols have varied greatly between laboratories and several have been optimized for large-scale clinical use despite associated complexity and high cost. Here, we present a simple method of "adherent" enrichment and expansion of NK cells, developed using both healthy donors' and cancer patients' peripheral blood mononuclear cells (PBMCs), and compare its effectiveness with various published protocols to highlight the pros and cons of their use in adoptive cell therapy. By building upon the concepts and data presented, future research can be adapted to provide simple, cost-effective, reproducible, and translatable procedures for personalized treatment with NK cells.

Irritable Bowel Syndrome: Yoga as Remedial Therapy.

Irritable bowel syndrome (IBS) is a group of symptoms manifesting as a functional gastrointestinal (GI) disorder in which patients experience abdominal pain, discomfort, and bloating that is often relieved with defecation. IBS is often associated with a host of secondary comorbidities such as anxiety, depression, headaches, and fatigue. In this review, we examined the basic principles of Pancha Kosha (five sheaths of human existence) concept from an Indian scripture Taittiriya Upanishad and the pathophysiology of a disease from the Yoga approach, Yoga Vasistha's Adhi (originated from mind) and Vyadhi (ailment/disease) concept. An analogy between the age old, the most profound concept of Adhi-Vyadhi, and modern scientific stress-induced dysregulation of brain-gut axis, as it relates to IBS that could pave way for impacting IBS, is emphasized. Based on these perspectives, a plausible Yoga module as a remedial therapy is provided to better manage the primary and secondary symptoms of IBS.

Hapten-induced contact hypersensitivity, autoimmune reactions, and tumor regression: plausibility of mediating antitumor immunity.

Haptens are small molecule irritants that bind to proteins and elicit an immune response. Haptens have been commonly used to study allergic contact dermatitis (ACD) using animal contact hypersensitivity (CHS) models. However, extensive research into contact hypersensitivity has offered a confusing and intriguing mechanism of allergic reactions occurring in the skin. The abilities of haptens to induce such reactions have been frequently utilized to study the mechanisms of inflammatory bowel disease (IBD) to induce autoimmune-like responses such as autoimmune hemolytic anemia and to elicit viral wart and tumor regression. Hapten-induced tumor regression has been studied since the mid-1900s and relies on four major concepts: (1) ex vivo haptenation, (2) in situ haptenation, (3) epifocal hapten application, and (4) antigen-hapten conjugate injection. Each of these approaches elicits unique responses in mice and humans. The present review attempts to provide a critical appraisal of the hapten-mediated tumor treatments and offers insights for future development of the field.

Augmentation of anti-HLA-E antibodies with concomitant HLA-Ia reactivity in IFNγ-treated autologous melanoma cell vaccine recipients.

HLA-E expressed on the surface of melanoma cells and shed into circulation are known to inhibit killing of tumor cells by binding to CD94/NKGA2 receptors on cytotoxic T- and NKT cells. Interferon (IFN)-γ is known to promote HLA-E over-expression on the cell surface and shedding. The shed HLA-E heavy chain may expose cryptic epitopes to elicit antibodies (Abs). The anti-HLA-E Abs may bind to shed HLA-E or to the tumor cell surface to block its interaction with CTL/NKT cells. This is the basis for a melanoma cell vaccine that will generate anti-HLA-E Abs. The objective of this study was to characterize the antibody response and characterize the cross-reactivity of the antibodies produced in melanoma patients immunized with autologous melanoma cells treated with IFNγ. Anti-HLA-E murine mAbs and serum anti-HLA-E Abs in healthy individuals were known to react with HLA-Ia alleles, which is attributed to the presence of peptide sequences shared between HLA-E and HLA-Ia. Therefore, pre- and post-immune (weeks 4 and 24) serum Abs reacting to both HLA-E and HLA-Ia alleles were measured by multiplex Luminex®-based immunoassay. To ascertain whether the reactivity of the serum Abs to HLA-Ia was due to anti-HLA-E Abs, the shared-peptides were used to inhibit anti-HLA-E and HLA-Ia reactivities. The level of anti-HLA-E IgG in sera has increased post-immunization from its pre-immune level. Concomitantly, the HLA-Ia reactivity of the sera was also augmented. The reactivity of both anti-HLA-E Abs and HLA-Ia were inhibited by the shared-peptides. The HLA-Ia reactivity of the anti-HLA-E Abs in patients' sera is similar to the HLA-Ia reactivity of the anti-HLA-E mAbs and anti-HLA-E Abs in normal sera. The results establish the immunogenicity of HLA-E and also ascertain that the HLA-Ia reactivity of the anti-HLA-E Abs is due to shared-peptide epitopes.