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Multiple myeloma (MM) is a hematologic malignancy that is characterized by the accumulation of abnormal plasma cells (PCs) in the bone marrow (BM). Patient outcome may be improved with BiTE (bispecific T-cell engager) molecules, which redirect T cells to lyse tumor cells. B-cell maturation antigen (BCMA) supports PC survival and is highly expressed on MM cells. A half-life extended anti-BCMA BiTE molecule (AMG 701) induced selective cytotoxicity against BCMA-expressing MM cells (average half-maximal effective concentration, 18.8 ± 14.8 pM), T-cell activation, and cytokine release in vitro. In a subcutaneous mouse xenograft model, at all doses tested, AMG 701 completely inhibited tumor formation (P < .001), as well as inhibited growth of established tumors (P ≤ .001) and extended survival in an orthotopic MM model (P ≤ .01). To evaluate AMG 701 bioactivity in cynomolgus monkeys, a PC surface phenotype and specific genes were defined to enable a quantitative digital droplet polymerase chain reaction assay (sensitivity, 0.1%). Dose-dependent pharmacokinetic and pharmacodynamic behavior was observed, with depletion of PC-specific genes reaching 93% in blood and 85% in BM. Combination with a programmed cell death protein 1 (PD-1)–blocking antibody significantly increased AMG 701 potency in vitro. A model of AMG 701 binding to BCMA and CD3 indicates that the distance between the T-cell and target cell membranes (ie, the immunological synapse) is similar to that of the major histocompatibility complex class I molecule binding to a T-cell receptor and suggests that the synapse would not be disrupted by the half-life extending Fc domain. These data support the clinical development of AMG 701.

Mast cells play key roles in allergy, anaphylaxis/anaphylactoid reactions, and defense against pathogens/toxins. These cells contain cytoplasmic granules with a wide spectrum of pleotropic mediators that are released upon activation. While mast cell degranulation (MCD) occurs upon clustering of the IgE receptor bound to IgE and antigen, MCD is also triggered through non-IgE-mediated mechanisms, one of which is via Mas-related G protein-coupled receptor X2 (MRGPRX2). MRGPRX2 can be activated by many basic biogenic amines and peptides. Consequently, MRGPRX2-mediated MCD is an important potential safety liability for peptide therapeutics. To facilitate peptide screening for this liability in early preclinical drug development, a rapid, high-throughput engineered CHO-K1 cell-based MRGPRX2 activation assay was evaluated and compared to histamine release in CD34+ stem cell-derived mature human mast cells as a reference assay, using 30 positive control and 29 negative control peptides for MCD. Both G protein-dependent (Ca2+ endpoint) and -independent (β-arrestin endpoint) pathways were assessed in the MRGPRX2 activation assay. The MRGPRX2 activation assay had a sensitivity of 100% for both Ca2+ and β-arrestin endpoints and a specificity of 93% (β-arrestin endpoint) and 83% (Ca2+ endpoint) compared to histamine release in CD34+ stem cell-derived mature human mast cells. These findings suggest that assessing MRGPRX2 activation in an engineered cell model can provide value as a rapid, high-throughput, economical mechanism-based screening tool for early MCD hazard identification during preclinical safety evaluation of peptide-based therapeutics.

Multiple myeloma (MM) is a cancer of the antibody-producing plasma cells (PC). MM is invariably fatal due to frequent disease relapse and/or treatment refractoriness, and therapies that provide deeper and more durable responses are needed. BiTE® (Bispecific T Cell Engager) molecules are immunotherapy agents that redirect a patient’s T cells to lyse tumor cells by simultaneously engaging a tumor associated antigen (TAA) on cancer cells and CD3ε on T cells. Clinical activity has been observed in MM using BiTE® molecules and other T cell engagers that target B-cell maturation antigen (BCMA). BCMA is an attractive MM TAA due to its broad prevalence and restricted normal tissue profile, mainly PCs and plasmablasts. However, heterogenous expression in MM cancer cells, potential antigen loss, and the presence of high levels of soluble BCMA in MM patient sera present challenges that may prevent BCMA-only-targeted therapies from achieving their full potential. To address these issues, we generated a BiTE® molecule capable of engaging BCMA and a second MM TAA, CS1 (also known as SLAMF7). This BiTE® molecule also contains an Fc-based domain to provide half-life extension (HLE). Here, we evaluated the in vitro and in vivo properties of this BCMA-CS1 HLE BiTE® molecule. The BCMA-CS1 HLE BiTE® molecule had nanomolar binding affinity for human BCMA, CS1, and CD3ε and the non-human primate (NHP) orthologues. In vitro, we observed picomolar activity against human MM cell lines with a range of BCMA and CS1 expression in a T cell dependent cellular cytotoxicity (TDCC) assay using human T cells or NHP peripheral blood mononuclear cells. The BCMA-CS1 HLE BiTE® molecule retained TDCC activity in the presence of soluble BCMA up to 2500 ng/mL as well as against human MM cells engineered to express only BCMA or CS1. In vivo, this BiTE® molecule inhibited tumor growth in a MM xenograft model. We also evaluated the BCMA-CS1 HLE BiTE® molecule in NHP over 15 days (IV dosing; intra-animal dose escalation from 60→240 μg/kg or 180→540 μg/kg on days 1 and 8). We observed hallmarks of BiTE® molecule activity in all groups, including transient decreases in circulating lymphocytes and moderate increases in cytokines like MCP-1. We measured the PC-specific transcripts BCMA and J-chain in NHP bone marrow and blood as surrogates for PC levels using ddPCR. These transcripts were reduced in both treatment groups (≥90%) with the strongest effects occurring in the 180→540 μg/kg group. Lastly, we showed that CS1 is highly and broadly expressed in MM patient samples and is restricted to a few normal hematopoietic cell types including PCs, NK cells, T cells, and some monocytes. These data suggest that the BCMA-CS1 HLE BiTE® molecule has potent in vitro and in vivo activity and may provide therapeutic benefit for MM patients by expanding the population of MM cancer cells that can be eliminated by a BiTE® molecule while overcoming common mechanisms that can impair BCMA-only-targeted MM therapies. Citation Format: Elizabeth T. Andrews, Stephanie C. Casey, Mohammad Farhad Amani, Grit Lorenczewski, Mozhgan Farshbaf, Lisa Winkel, Matthias Klinger, John M. Harrold, Famke Aeffner, Ana Goyos, Matthias Friedrich, Tara Arvedson, Matthew G. Chun. Evaluation of a dual-targeting BCMA-CS1 HLE BiTE® molecule for multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6251.

Natural killer (NK) cells are lymphocytes capable of cytotoxicity against virally infected cells and tumor cells. The display of effector function by NK cells is the result of interactions between germline encoded activating/inhibitory NK cell receptors and their ligands (major histocompatibility complex class I, major histocompatibility complex class I-like, viral, and cellular stress-related surface molecules) expressed on target cells. Determination of NK cell number and function is a common element of the immunotoxicology assessment paradigm for the development of certain classes of pharmaceuticals across a range of modalities. This article summarizes the evidence associating NK cell dysfunction with infectious and cancer risks, reviews emerging NK cell biology, including the impact of immunogenetics on NK cell education and function, and provides perspectives about points to consider when assessing NK cell function in different species in the context of safety assessment.

Chimpanzees have orthologs of the six fixed, functional human MHC class I genes. But, in addition, the chimpanzee has a seventh functional gene, Patr-AL, which is not polymorphic but contributes substantially to population diversity by its presence on only 50% of MHC haplotypes. The ancestral AL gene emerged long before the separation of human and chimpanzee ancestors and then subsequently and specifically lost function during human evolution, but was maintained in chimpanzees. Patr-AL is an alloantigen that participates in negative and positive selection of the T cell repertoire. The three-dimensional structure and the peptide-binding repertoire of Patr-AL and HLA-A*02 are surprisingly similar. In contrast, the expression of these two molecules is very different, as shown using specific mAbs and polyclonal Abs made against Patr-AL. Peripheral blood cells and B cell lines express low levels of Patr-AL at the cell surface. Higher levels are seen for 221-cell transfectants expressing Patr-AL, but in these cells a large majority of Patr-AL molecules are retained in the early compartments of the secretory pathway: mainly the endoplasmic reticulum, but also cis-Golgi. Replacing the cytoplasmic tail of Patr-AL with that of HLA-A*02 increased the cell-surface expression of Patr-AL substantially. Four substitutions distinguish the Patr-AL and HLA-A*02 cytoplasmic tails. Systematic mutagenesis showed that each substitution contributes changes in cell-surface expression. The combination of residues present in Patr-AL appears unique, but each individual residue is present in other primate MHC class I molecules, notably MHC-E, the most ancient of the functional human MHC class I molecules.

HLA-B∗46:01 was formed by an intergenic mini-conversion, between HLA-B∗15:01 and HLA-C∗01:02, in Southeast Asia during the last 50,000 years, and it has since become the most common HLA-B allele in the region. A functional effect of the mini-conversion was introduction of the C1 epitope into HLA-B∗46:01, making it an exceptional HLA-B allotype that is recognized by the C1-specific natural killer (NK) cell receptor KIR2DL3. High-resolution mass spectrometry showed that HLA-B∗46:01 has a low-diversity peptidome that is distinct from those of its parents. A minority (21%) of HLA-B∗46:01 peptides, with common C-terminal characteristics, form ligands for KIR2DL3. The HLA-B∗46:01 peptidome is predicted to be enriched for peptide antigens derived from Mycobacterium leprae. Overall, the results indicate that the distinctive peptidome and functions of HLA-B∗46:01 provide carriers with resistance to leprosy, which drove its rapid rise in frequency in Southeast Asia. Fil: Hilton, Hugo G.. University of Stanford; Estados Unidos Fil: McMurtrey, Curtis P.. Oklahoma State University; Estados Unidos Fil: Han, Alex S.. University of Stanford; Estados Unidos Fil: Djaoud, Zakia. University of Stanford; Estados Unidos Fil: Guethlein, Lisbeth A.. University of Stanford; Estados Unidos Fil: Blokhuis, Jeroen H.. University of Stanford; Estados Unidos Fil: Pugh, Jason L.. University of Stanford; Estados Unidos Fil: Goyos, Ana. University of Stanford; Estados Unidos Fil: Horowitz, Amir. University of Stanford; Estados Unidos Fil: Buchli, Rico. Pure Protein LLC; Estados Unidos Fil: Jackson, Ken W.. Oklahoma State University; Estados Unidos Fil: Bardet, Wilfred. Oklahoma State University; Estados Unidos Fil: Bushnell, David A.. University of Stanford; Estados Unidos Fil: Robinson, Philip J.. University of Stanford; Estados Unidos Fil: Mendoza, Juan L. University of Stanford; Estados Unidos Fil: Birnbaum, Michael E.. University of Stanford; Estados Unidos Fil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Garcia, K. Christopher. University of Stanford; Estados Unidos Fil: Hildebrand, William H.. Oklahoma State University; Estados Unidos Fil: Parham, Peter. University of Stanford; Estados Unidos

CD4 and CD8 co-receptors play critical roles in T cell development and activation by interacting both with T cell receptors and MHC molecules. Although homologs of these genes have been identified in many jawed vertebrates, there are still unresolved gaps concerning their evolution and specialization in MHC interaction and T cell function. Using experimental and computational procedures we identified CD4, CD8α and CD8β gene homologs both in Xenopus tropicalis, whose full genome has been sequenced, and its sister species Xenopus laevis. Multiple alignments of deduced amino acid sequences reveal a poor conservation of the residues involved in binding of CD4 to MHC class II, and CD8α to class I in non-mammalian species, presumably related to the co-evolutionary pressure of MHC I and II genes. Phylogenetic study suggests that Xenopodinae co-receptor genes are more closely related to their homologs in other tetrapods than those of bony fish. Furthermore, the developmental and cell-specific expression patterns of these genes in X. laevis are very similar to that of mammals. X. laevis CD4 is mainly expressed by peripheral non-CD8 T cells and detected in the thymus as early as four days post-fertilization (dpf) at the onset of thymic organogenesis. CD8β expression is specific to adult surface CD8(+) T cells and thymocytes, and is first detected in the thymus at 5 dpf in parallel with productive TCRγ transrcipts, whereas productive TCRβ and α rearrangements are not detected before 7-9 dpf.

Nonclassical MHC class Ib (class Ib) genes are heterogeneous genes encoding molecules that are structurally similar to classical MHC class Ia molecules but with limited tissue distribution and polymorphism. Mammalian class Ib genes have diverse and often uncharacterized functions, and because of their rapid rate of evolution, class Ib phylogeny is difficult to establish. We have conducted an extensive genomic, molecular, and phylogenetic characterization of class Ib genes in two Xenopodinae amphibian species of different genera that diverged from a common ancestor as long ago as primates and rodents (∼65 million years). In contrast with the unsteadiness of mammalian class Ib genes, our results reveal an unusual degree of conservation of most Xenopodinae class Ib gene lineages, including a novel monogenic lineage represented by the divergent Xenopus laevis XNC10 gene and its unequivocal Silurana (Xenopus) tropicalis orthologue, SNC10. The preferential expression of this gene lineage by thymocytes themselves from the onset of thymic organogenesis is consistent with a specialized role of class Ib in early T cell development and suggests such a function is conserved in all tetrapods.

Background: Multiple myeloma (MM) is a hematologic malignancy where abnormal plasma cells (PC) accumulate in the bone marrow (BM). Patients often develop relapsed/refractory disease leading to significant morbidity and mortality. Elimination of residual disease is required for curative treatments. Patient outcome can be improved by using T-cell engaging agents, such as bispecific T-cell engager (BiTE®) antibody constructs, to redirect T cells to tumor cells. B-cell maturation antigen (BCMA), a TNF receptor superfamily member, supports PC survival and is widely expressed on MM and normal PC. Methods: A half-life extended (HLE) anti-BCMA BiTE® (BCMA HLE-BiTE®) antibody construct was generated and characterized in vitro, and in vivo in a mouse xenograft model and in non-human primates (NHP). To quantify on-target activity of the BCMA HLE-BiTE® in NHP it was necessary to follow changes in PC numbers. Because PC are rare (0.1 - 3%) in the BM and blood, detection using traditional assays is not possible. To address this limitation, a PC gene signature was defined and used to develop a quantitative ddPCR assay (sensitivity = 0.01%) to assess the effect of the BCMA HLE-BiTE® in a 12-day repeat-dose NHP study. Results: A BCMA HLE-BiTE® was selected that demonstrated cytotoxic activity using human effector cells (EC50: 0.13 ± 0.07 pM in MM.1R cells) and comparable activity using NHP effector cells. In a sub-cutaneous mouse xenograft model in which BCMA-positive NCI-H929 cells and human PBMC were co-administered, the BCMA HLE-BiTE® completely inhibited tumor formation, in contrast to vehicle controls with/without PBMC. The serum half-life of the BCMA HLE-BiTE® in NHP was 112 hours following a single 15 µg/kg IV dose. The PK/PD relationship in NHP was next evaluated in a 12-day repeat-dose study. A dose-dependent PK behavior as well as depletion of PC in blood and BM was observed, reaching 93% depletion in the blood and 85% in the BM in the high-dose group on day 12. Conclusions: These results demonstrate that the BCMA HLE-BiTE® is potent and effective at inducing cytotoxicity of target cells in vitro and in vivo, and may be suitable for once-weekly dosing in MM patients. This molecule is being evaluated in humans: trial NCT03287908. Citation Format: Ana Goyos, Chi-Ming Li, Petra Deegen, Pamela Bogner, Oliver Thomas, Joachim Wahl, Rebecca Goldstein, Maththias Friedrich, Angela Coxon, Mercedesz Balazs, Tara Arvedson. Cynomolgus monkey plasma cell gene signature to quantify the in vivo activity of a half-life extended anti-BCMA BiTE® for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-299.

In jawed vertebrates, the heterogeneous nonclassical MHC class Ib (class Ib) gene family encodes molecules structurally similar to classical MHC class Ia (class Ia) but with more limited tissue distribution and lower polymorphism. In mammals, class Ib gene products are involved in stress responses, malignancy and differentiation of intrathymic CD8 T cells. The frog Xenopus laevis possesses at least 20 class Ib genes (XNCs), and 9 subfamilies have been defined so far. We have characterized two novel subfamilies, XNC10 and XNC11. XNC10 is phylogenetically and structurally distinct from both class Ia and other XNC genes. Besides thymic lymphoid tumors, XNC10 is preferentially expressed by circulating T cells and thymocytes of the CD8 lineage both in adult and in larvae from the onset of thymus organogenesis. XNC11 is expressed only by thymocytes and upregulated by several thymic lymphoid tumors. These data provide the first evidence of the expression of any class Ib genes in Xenopus larvae, and suggests evolutionary relationships between certain class Ib genes, malignancy and CD8 T cell ontogeny.

The heat shock proteins (HSPs) gp96 and hsp70 can elicit potent anti-tumor responses and as such have significant clinical potential. Besides cytotoxic CD8 T cell (CTLs) effectors, evidence suggests that natural killer (NK) cells and other less well-characterized cell types also play a critical role in HSP-mediated anti-tumor responses. Owing to their high degree of phylogenetic conservation, we have proposed that HSPs are ancestral agents of immune surveillance; and postulated that their immunological properties, if important, should have been conserved during evolution. We are investigating this issue using a unique non-mammalian comparative tumor-immunity model in the frog Xenopus, which allows us to focus on the relationship between HSPs, classical MHC class Ia, and non-classical MHC class Ib molecules. In addition to a transplantable lymphoid tumor in genetically defined cloned Xenopus, we are generating transgenic frogs with inducible or knocked-down (RNAi) gene expression.

Modulating natural killer cell functions in human immunity and reproduction are diverse interactions between the killer cell immunoglobulin-like receptors (KIR) of Natural Killer (NK) cells and HLA class I ligands on the surface of tissue cells. Dominant interactions are between KIR2DL1 and the C2 epitope of HLA-C and between KIR2DL2/3 and the C1 epitope of HLA-C. KhoeSan hunter-gatherers of Southern Africa represent the earliest population divergence known and are the most genetically diverse indigenous people, qualities reflected in their KIR and HLA genes. Of the ten KhoeSan KIR2DL1 alleles, KIR2DL1*022 and KIR2DL1*026 likely originated in the KhoeSan, and later were transmitted at low frequency to the neighboring Zulus through gene flow. These alleles arose by point mutation from other KhoeSan KIR2DL1 alleles that are more widespread globally. Mutation of KIR2DL1*001 gave rise to KIR2DL1*022, causing loss of C2 recognition and gain of C1 recognition. This makes KIR2DL1*022 a more avid and specific C1 receptor than any KIR2DL2/3 allotype. Mutation of KIR2DL1*012 gave rise to KIR2DL1*026, causing premature termination of translation at the end of the transmembrane domain. This makes KIR2DL1*026 a membrane-associated receptor that lacks both a cytoplasmic tail and signaling function. At higher frequencies than their parental allotypes, the combined effect of the KhoeSan-specific KIR2DL1*022 and KIR2DL1*026 is to reduce the frequency of strong inhibitory C2 receptors and increase the frequency of strong inhibitory C1 receptors. Because interaction of KIR2DL1 with C2 is associated with risk of pregnancy disorder, these functional changes are potentially advantageous. Whereas all other KhoeSan KIR2DL1 alleles are present on a wide diversity of centromeric KIR haplotypes, KIR2DL1*026 is present on a single KIR haplotype and KIR2DL1*022 is present on two very similar haplotypes. The high linkage disequilibrium across their haplotypes is consistent with a recent emergence for these KIR2DL1 alleles that have distinctive functions., Author Summary The genes that control the response of the human immune system vary enormously between individuals. Understanding the evolution of these genetic differences and how they individualize immune responses is central to understanding how the immune system works in health and disease. In this regard, the KhoeSan of southern Africa are particularly informative because they are genetically diverse, divergent from other modern human populations and have been subject to unique demographic history. In the KhoeSan population, we studied variable genes that control natural killer cell function. We identified two recently evolved, novel gene variants that have unusual function; one completely changed its ligand specificity and the other lost its capacity for signal transduction.

In Xenopus as in mammals, gp96 stimulates MHC-restricted cellular immunity against chaperoned minor histocompatibility (H) antigens (Ag). In adult Xenopus, gp96 also elicits peptide-specific effectors against MHC class Ia-negative 15/0 tumors. To determine whether gp96 can generate functionally heterogeneous CD8+ effectors (CTL that kill MHC class Ia+ minor H-Ag-disparate lymphoblasts and MHC class Ia- tumor targets), LG-6 isogenetic frogs were immunized with gp96 purified either from MHC-identical but minor H-Ag-disparate LG-15 normal tissues or from the MHC class Ia-negative 15/0 tumor line (derived from LG-15 frogs). LG-15 normal liver-derived gp96 did not induce detectable CD8+ in vitro killing against 15/0 tumor cells. However, 15/0-derived gp96 did induce killing against both MHC class Ia+ LG-15 lymphoblasts and the MHC class Ia- 15/0 tumor, but not against another MHC class Ia- tumor (B3B7) or against LG-6 lymphoblasts. Tumor killing was better when 15/0 rather than normal LG-15 irradiated stimulators were used, but in vitro stimulation without prior in vivo immunization was ineffective. These data suggest that (1) 15/0-derived gp96 chaperones minor H-Ag shared with normal LG-15 lymphocytes and elicits MHC-restricted CTL, and (2) 15/0-derived gp96, but not normal liver-derived gp96, generates CD8+ effectors that kill 15/0 tumor cells in the absence of MHC class Ia expression.

Nonclassical MHC class Ib (class Ib) genes are a family of highly diverse and rapidly evolving genes wherein gene numbers, organization, and expression markedly differ even among closely related species rendering class Ib phylogeny difficult to establish. Whereas among mammals there are few unambiguous class Ib gene orthologs, different amphibian species belonging to the anuran subfamily Xenopodinae exhibit an unusually high degree of conservation among multiple class Ib gene lineages. Comparative genomic analysis of class Ib gene loci of two divergent (~65 million years) Xenopodinae subfamily members Xenopus laevis (allotetraploid) and Xenopus tropicalis (diploid) shows that both species possess a large cluster of class Ib genes denoted as Xenopus/Silurana nonclassical (XNC/SNC). Our study reveals two distinct phylogenetic patterns among these genes: some gene lineages display a high degree of flexibility, as demonstrated by species-specific expansion and contractions, whereas other class Ib gene lineages have been maintained as monogenic subfamilies with very few changes in their nucleotide sequence across divergent species. In this second category, we further investigated the XNC/SNC10 gene lineage that in X. laevis is required for the development of a distinct semi-invariant T cell population. We report compelling evidence of the remarkable high degree of conservation of this gene lineage that is present in all 12 species of the Xenopodinae examined, including species with different degrees of ploidy ranging from 2, 4, 8 to 12 N. This suggests that the critical role of XNC10 during early T cell development is conserved in amphibians.

Despite intense study, the role of the immune system in detecting (immunosurveillance), controlling and remodeling (immunoediting) neoplasia remains elusive. We present here a comparative view of the complex interactions between neoplasia and the host immune system. We provide evidence, in the amphibian Xenopus laevis, consistent with an evolutionarily conserved and crucial role of the immune system in controlling neoplasia, which involves a striking variety of anti-tumoral immune effectors including conventional CTLs, classical MHC class Ia unrestricted CTLs (CCU-CTLs) that interact with nonclassical MHC class Ib molecules, CD8 NKT-like cells and NK cells. We also review the tumors found in X. laevis with an emphasis on thymic lymphoid tumors and a rare ovarian dysgerminoma. Finally, we consider the use of X. laevis for in vivo study of tumorigenesis. Given our current knowledge, the experimental systems already established in X. laevis, and the rapid accumulation of genetic resources for the sister species Silurana (Xenopus) tropicalis, it is our conviction that these species provide an ideal alternative to the murine system for studying tumorigenesis and tumor immunity.

The image of the jumping frog was kindly provided by Jacques Robert and created by his son by Adrien. Anti-tumor responses, both in vitro and in vivo, are studied using Xenopus laevis by Robert and colleagues (pp. 1494–1501); non-classical MHC class Ib molecules are shown, by RNA interference, to be important for the gp96-induced CTL response against class Ia-negative tumors in Xenopus. The conservation of the class Ia-unrestricted response from Xenopus to mammals indicates the importance of this anti-tumor immune mechanism.

Nonclassical MHC class Ib (class Ib) genes are found in all jawed vertebrates, and their products are hypothesized to be indicators of intracellular stress and malignancy. They may be involved in immune recognition of classical MHC class Ia (class Ia)-low or -negative tumor cells through their interaction with T cell receptors and/or non-T cell inhibitory or triggering receptors expressed by NK cells and T cells. In the frog Xenopus, the molecular chaperone gp96 mediates a potent immune response involving antigen-specific classical class Ia-unrestricted CD8+ CTL (CCU-CTL) against a transplantable thymic tumor (15/0) that does not express class Ia molecules. We hypothesized that Xenopus nonclassical class Ib gene products (XNC) are involved in gp96-mediated CCU-CTL anti-tumor responses. To investigate the involvement of class Ib gene products in Xenopus anti-tumor responses, we generated, for the first time in ectothermic vertebrates, stable tumor transfectants expressing short hairpin RNA (shRNA) to silence either XNC directly or beta2m to prevent class Ib surface expression. Both types of 15/0 transfectants are more resistant to CCU-CTL killing, more tumorigenic and more susceptible to NK-like cell killing. This study provides in vitro and in vivo evidence of the evolutionary conservation of class Ib involvement in anti-tumor CD8+ T cell responses.

Aim The click-speaking Khoesan of Southern Africa represent the oldest living lineage and are among the most genetically diverse, of modern human populations. In studying the extent and evolution of human immunogenetic diversity, strong balancing selection has been identified in the Khoesan HLA region. The aim of our study was to investigate the genetic and functional diversity of HLA-C and KIR interactions in the Khoesan and compare them to populations with different demographic and genetic histories. Methods We investigated the allelic diversity of KIR2DL1, 2DL2, 2DL3 (using pyrosequencing) and HLA-C (using Luminex) in 56 unrelated Khoesan hunter-gatherers from Upington, South Africa. To measure ligand-specificity and strength, KIR-Fc fusion proteins were made for each identified allotype and tested against One Lambda Labscreen HLA class I beads. To measure the population-wide combinatorial reactivity of KIR and HLA allotypes we devised a scoring system that accounted for their surface expression, binding and signaling capacity as well as their frequencies. Results We identified two novel alleles of KIR2DL1, which are common in the Khoesan. Analysis of 1000 genomes data showed these alleles are unique to this population. 2DL1 ∗ 022 encodes lysine at position 44 which changes its specificity from HLA-C2 to C1 and 2DL1 ∗ 026 binds HLA-C2 but does not transduce an inhibitory signal because it lacks a cytoplasmic tail. Together these alleles reduce the capacity of 2DL1 to function as a C2 receptor in the Khoesan. When accounting for functional characteristics of receptor/ligand pairs, the population wide reactivity of 2DL1-C2 was much lower in the Khoesan than predicted from allele frequencies alone. In contrast, the modeled reactivity of 2DL3-C1 was unaffected. We observe an inverse correlation between functionally interactive allotypes and ligand frequencies such that combinatorial reactivity is equilibrated across populations. Conclusions The Khoesan have a diverse and unique repertoire of KIR genes that have evolved under strong selective pressure from reproductive and infectious disease. Although allele frequency spectra of both loci differ significantly, the signature of these selective pressures remains in the form of KIR and HLA functional profiles that are equilibrated between human populations.

CD91, the endocytic receptor for alpha2-macroglobulin (alpha2M), mediates the internalization of certain heat shock proteins (hsps) and the cross-presentation of peptides they chaperone by antigen-presenting cells. The phylogenetic conservation of the immunologically active CD91 ligands, alpha2M and hsps, is consistent with the idea of an ancestral system of immune surveillance. We have further explored this hypothesis by taking advantage of the frog Xenopus, and asked how conserved is CD91 and whether the expression of CD91 is differentially modulated during immune responses of class I-positive adult and naturally class I-negative larvae. We have identified a Xenopus CD91 gene homologue that displays high sequence identity (65%) with other CD91 homologues and contains an additional distinctive cytoplasmic NPXY motif. Phylogenetic analysis indicates that CD91 homologues branch as a monophyletic group distinct from other LDLRs; this suggests an origin of CD91 contemporary with that of metazoans. A 14-kb transcript is detected by Northern blotting in most adult and larval tissues, including lymphoid tissues. RT-PCR study reveals that CD91 is expressed in most cell types, including adult macrophages, B and T cells as well as in splenocytes and thymocytes from naturally MHC class I negative larvae. CD91 is markedly up-regulated in vivo by adult peritoneal leukocytes following bacterial and viral stimulation; it is constitutively expressed on class I-negative larval peritoneal leukocytes at high levels and cannot be further upregulated by such stimulation. These data are in agreement with a conserved role of CD91 in immunity.

Although CD4 and CD8 gene homologs have been identified in many jawed vertebrates, phylogeny and functional conservation of these molecules is not fully understood. We have characterized CD4, CD8α and CD8β gene homologs in two species of the subfamily Xenopodinae, S. tropicalis, whose full genome is sequenced, and X. laevis. Multiple alignments of amino acid sequences, in these two species, as in other nonmammalian species, reveal a poor conservation of the residues of CD4 and CD8α that bind to MHC class II and class I molecules, respectively, suggesting species-specific coevolution. Phylogenetic analysis indicates that all three co-receptor genes are more related to other tetrapods than to bony fish. Furthermore, developmental and cell-specific expression patterns of these genes in X. laevis are very similar to that of mammals. X. laevis CD4 is mainly expressed by peripheral non-CD8 T cells and detected in the thymus at the onset of organogenesis, 5 days post-fertilization (dpf). The CD8β gene is specifically expressed by adult CD8 positive T cells and thymocytes, and is first detected in the thymus and intestine at 6 dpf. TCRα and TCRγ transcripts are also detected at 6 dpf, before TCRβ (9 dpf). TCRγ transcripts cloned from 7dpf are fully rearranged and in frame. This suggests that T cell subsets, possibly γ/δ T cells or even α/β T cells, reside in the intestine before the onset of thymocyte differentiation, and may contribute to mucosal immunity early in development.

The heat shock proteins (hsps) gp96 and hsp70 mediate potent antigen-dependent anti-tumor responses in both mammals and Xenopus. We have developed an adoptive cell transfer assay using peritoneal leukocytes as antigen presenting cells (APCs), and shown that hsp72, but not hsc73, is as potent as gp96 to prime T cell responses in vivo against the Xenopus thymic tumor 15/0 that is tumorigenic when transplanted in MHC-compatible Xenopus clones. The 15/0 tumor expresses several nonclassical MHC class Ib genes and β2-m, however it does not express classical MHC class Ia. Despite the lack of class Ia expression by 15/0, Xenopus adults generate potent cytotoxic CD8 T cell effectors (CCU-CTLs) that specifically recognize and kill the 15/0 tumor. We postulate that hsp72, but not hsc73, can prime class Ib-mediated anti-tumor CCU-CTL responses by interacting with APCs. To reveal the roles of class Ia and class Ib in this process we silenced surface expression of both molecules on APCs and assessed the effects in priming hsp72- or hsc73-mediated tumor immunity by adoptive transfer. To further elucidate the involvement of class Ia and class Ib in hsp70-mediated T cell function in vivo, we developed reliable transgenic techniques for our Xenopus clones and obtained F0 animals with silenced β2-m expression in vivo. This study allows us to gain better perspective into the intricate relationship between the two hsps, class Ia and class Ib molecules in T cell function as well as tumor immunity.

In mammals, certain heat shock proteins (hsps) participate in specialized responses to stressors associated with pathogens or tumors, and as such, act as agents of immune surveillance interacting with both innate and adaptive immunity. We are investigating the conservation of this role throughout the class of vertebrates. We have shown that in Xenopus as in mammals, gp96, the major resident of the endoplasmic reticulum, generates MHC-restricted thymus-dependent immunity in vivo and CR in vitro against minor histocompatibility (H) antigens. By as yet unclear mechanisms that may involve classical MHC-unrestricted cytotoxic CD8+ T cells, gp96 also elicits peptide-specific responses against MHC-class I-negative tumors in adult frogs that may involve cytotoxic NK, MHC-unrestricted CD8+ T and NK/T cells. In naturally MHC class I-deficient but immunocompetent Xenopus larvae, gp96 also generates an innate type of anti-tumor response that is independent of chaperoned peptides. Finally, in a subset of Xenopus sIgM+ B cells, a substantial fraction of gp96 is directed to the cell surface by an active process that is upregulated by bacterial stimulation. This may allow gp96 to access the extracellular compartment without necrosis. Given the dual abilities of gp96 to chaperone antigenic peptides and to modulate innate immune responses, we propose that stimulated B cells that are up-regulating surface gp96 can directly interact with antigen presenting cells (APC) and/or T helper (Th) cells to trigger or amplify immune responses.