PD-1-specific "Blocking" antibodies that deplete PD-1+ T cells present an inconvenient variable in preclinical immunotherapy experiments.

Therapeutic antibodies blocking PD-1-/PD-L1 interaction have achieved remarkable clinical success in cancer. In addition to blocking a target molecule, some isotypes of antibodies can activate complement, NK cells or phagocytes, resulting in death of the cell expressing the antibody's target. Human anti-PD-1 therapeutics use antibody isotypes designed to minimize such antibody-dependent lysis. In contrast, anti-PD-1 reagents used in mice are derived from multiple species, with different isotypes, and are not engineered to reduce target cell death: few studies analyze or discuss how antibody species and isotype may impact data interpretation. We demonstrate here that anti-PD-1 therapy to promote activation and proliferation of murine PD-1-expressing CD8 T cells sometimes led instead to a loss of antigen specific cells. This phenomenon was seen in two tumor models and a model of virus infection, and varied with the clone of anti-PD-1 antibody. Additionally, we compared competition among anti-PD-1 clones to find a combination that allows detection of PD-1-expressing cells despite the presence of blocking anti-PD1 antibodies in vivo. These data bring attention to the possibility of unintended target cell depletion with some commonly used anti-mouse PD-1 clones, and should provide a valuable resource for the design and interpretation of anti-PD-1 studies in mice.

Allergome-wide peptide microarrays enable epitope deconvolution in allergen-specific immunotherapy.

BACKGROUND: The interaction of allergens and allergen-specific IgE initiates the allergic cascade after crosslinking of receptors on effector cells. Antibodies of other isotypes may modulate such a reaction. Receptor crosslinking requires binding of antibodies to multiple epitopes on the allergen. Limited information is available on the complexity of the epitope structure of most allergens. OBJECTIVES: We sought to allow description of the complexity of IgE, IgG4, and IgG epitope recognition at a global, allergome-wide level during allergen-specific immunotherapy (AIT). METHODS: We generated an allergome-wide microarray comprising 731 allergens in the form of more than 172,000 overlapping 16-mer peptides. Allergen recognition by IgE, IgG4, and IgG was examined in serum samples collected from subjects undergoing AIT against pollen allergy. RESULTS: Extensive induction of linear peptide-specific Phl p 1- and Bet v 1-specific humoral immunity was demonstrated in subjects undergoing a 3-year-long AIT against grass and birch pollen allergy, respectively. Epitope profiles differed between subjects but were largely established already after 1 year of AIT, suggesting that dominant allergen-specific antibody clones remained as important contributors to humoral immunity following their initial establishment during the early phase of AIT. Complex, subject-specific patterns of allergen isoform and group cross-reactivities in the repertoires were observed, patterns that may indicate different levels of protection against different allergen sources. CONCLUSIONS: The study highlights the complexity and subject-specific nature of allergen epitopes recognized following AIT. We envisage that epitope deconvolution will be an important aspect of future efforts to describe and analyze the outcomes of AIT in a personalized manner.

Human IgG subclass cross-species reactivity to mouse and cynomolgus monkey Fcγ receptors.

In therapeutic antibody discovery and early development, mice and cynomolgus monkey are used as animal models to assess toxicity, efficacy and other properties of candidate molecules. As more candidate antibodies are based on human immunoglobulin (IgG) subclasses, many strategies are pursued to simulate the human system in the test animal. However, translation rate from a successful preclinical trial to an approved drug is extremely low. This may partly be due to differences in interaction of human IgG based candidate molecules to endogenous Fcγ receptors of model animals in comparison to those of human Fcγ receptors. In this study, we compare binding characteristics of human IgG subclasses commonly used in drug development (IgG1, IgG2, IgG4) and their respective Fc silent versions (IgG1σ, IgG2σ, IgG4 PAA) to human, mouse, and cynomolgus monkey Fcγ receptors. To control interactions between Fab and Fc domains, the test IgGs all have the same variable region sequences. We found distinct variations of interaction of human IgG subclasses to model animal Fcγ receptors in comparison to their human counterparts. Particularly, cynomolgus monkey Fcγ receptors showed consistently tighter binding to human IgGs than human Fcγ receptors. Moreover, the presumably Fc silent human IgG4 PAA framework bound to cynomolgus monkey FcγRI with nanomolar affinity while only very weak binding was observed for the human FcγRI. Our results highlighted the need for a thorough in vitro affinity characterization of candidate IgGs against model animal Fcγ receptors and careful design of preclinical studies.

Effect of bovine colostrum feeding in comparison with milk replacer and natural feeding on the immune responses and colonisation of enterotoxigenic Escherichia coli in the intestinal tissue of piglets.

The present study investigated the effect of feeding bovine colostrum (BC) to piglets in comparison with feeding a milk replacer (MR) and conventional rearing by the sow on the intestinal immune system and number of enterotoxigenic Escherichia coli (ETEC) colonising the intestinal tissue. Piglets (23-d-old) were allocated to one of the following four groups: (1) killed at the beginning of the experiment (Base); (2) separated from the sow and fed BC (BC-fed); (3) separated from the sow and fed a MR (MR-fed); (4) kept with the sow (Sow-Milk). Blood was sampled on days 1 and 8, and faecal samples were collected on days 1, 3, 5 and 8. On day 8, piglets were killed and gastrointestinal digesta and intestinal segments were collected. The frequency of diarrhoea was found to be higher (P≤ 0·019) in MR-fed piglets than in BC-fed and Sow-Milk piglets. Piglets from the MR-fed group had the lowest lactic acid bacteria:haemolytic E. coli ratio (P(treat)= 0·064) in the faeces. The number of E. coli colonising the intestinal tissue was higher (P< 0·001) in piglets from the MR-fed group than in those from the BC-fed and Sow-Milk groups. Piglets from the Sow-Milk group had a higher (P= 0·020) mucosal IgG concentration than those from the MR-fed group, but did not exhibit any difference when compared with piglets from the Base and BC-fed groups. Piglets from the BC-fed group exhibited a reduced (P≤ 0·037) expression level of Toll-like receptor-4 in the intestinal mucosa when compared with those from the MR-fed and Sow-Milk groups. The expression level of IL-2 was higher (P≤ 0·051) in piglets from the MR-fed group than in those from the other treatment groups. In conclusion, feeding BC rather than MR to the piglets reduced the colonisation of intestine by ETEC and modulated the intestinal immune system, whereas no differences were observed in piglets fed BC and conventionally reared by the sows.

Toxicological effects of nickel chloride on IgA+ B Cells and sIgA, IgA, IgG, IgM in the intestinal mucosal immunity in broilers.

The objective of this study was to investigate the toxicological effects of dietary NiCl2 on IgA+ B cells and the immunoglobulins including sIgA, IgA, IgG and IgM in the small intestine and cecal tonsil of broilers by the methods of immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Two hundred and forty one-day-old avian broilers were randomly divided into four groups and fed on a control diet and three experimental diets supplemented with 300, 600, and 900 mg/kg NiCl2 for 42 days. Compared with the control group, the IgA+ B cell number and the sIgA, IgA, IgG, and IgM contents in the NiCl2-treated groups were significantly decreased (p < 0.05 or p < 0.01). It was concluded that dietary NiCl2 in the excess of 300 mg/kg had negative effects on the IgA+ B cell number and the above mentioned immunoglobulin contents in the small intestine and the cecal tonsil. NiCl2-reduced sIgA, IgA, IgG and IgM contents is due to decrease in the population and/or the activation of B cell. The results suggest that NiCl2 at high levels has intestinal mucosal humoral immunotoxicity in animals.

Maternal anemia induces changes in immunological and nutritional components of breast milk.

OBJECTIVE: The effects of low maternal hemoglobin levels on the immunological and nutritional components of breast milk at different maturation stages were investigated. METHODS: Colostrum, transitional and mature milk were collected from 25 mothers with normal hemoglobin levels (control group) and 18 mothers with hemoglobin levels below 11 g/dL (anemia group). Total protein, antibodies, complement proteins, fat and calorie, lipase, iron, transferrin levels, total iron-binding capacity, latent iron-binding capacity (LIBC) and transferrin saturation index (TSI) were determined. RESULTS: In contrast to the control group, anemic mothers had higher total protein levels in milk, lower IgA and IgG levels in colostrum, lower C3 protein levels in milk, lower C4 protein levels in colostrum and transitional milk, higher fat in the colostrum and lower calorie content in mature milk. In both groups, lipase was lower in mature milk and iron concentration was similar. Transitional and mature milk from anemic mothers had higher LIBC and lower TSI values. CONCLUSION: A decrease in maternal hemoglobin levels causes immunological and nutritional alterations in milk at different maturation stages. Special measures must therefore be taken for mothers at risk of developing anemia to ensure they can provide high-quality milk to their babies.

For the assessment of intestinal permeability, size matters.

The purpose of this review is to demonstrate that an intestine leaky to small molecules can be impermeable to large antigenic molecules. The author proposes that the permeability of the epithelium to very small sugar molecules such as lactulose/mannitol-used for the past 50 years to gauge intestinal permeability-does not necessarily correlate with epithelial permeability to macromolecules. This article begins with the history and science behind the use of small sugars to measure permeability, a method developed in 1899. The lactulose/mannitol test may give useful information regarding the overall condition of the digestive tract; however, the author suggests that the test is not indicative of the transport of macromolecules such as bacterial toxins and food antigens, which have the capacity to damage the structure of the intestinal barrier and/or challenge the immune system. This article describes the various mechanisms and physiological transport pathways through which increased antigen uptake may result in immunological reactions to food antigens and bacterial lipopolysaccharides, resulting in the pathogenesis of disease. Finally, the article presents evidence indicating that increased intestinal, antigenic permeability plays a key role in the development of various inflammatory and autoimmune disorders. Therefore, more knowledge about the epithelium's permeability to large molecules undoubtedly contributes not only to early detection but also to secondary prevention of many inflammatory autoimmune, neuroimmune, and neurodegenerative disorders.

Surrogate approaches in development of monoclonal antibodies.

When cross-reactivity of a lead antibody across species is limited, antibody development programs require the generation of surrogate molecules or surrogate animal models necessary for the conduct of preclinical pharmacology and safety studies. When surrogate approaches are employed, the complexities and challenges for translation of preclinical safety and efficacy results to the clinic are undoubtedly enhanced. Because there are no currently established criteria or regulatory guidance regarding the application of surrogate approaches, a science-based strategy for translation of preclinical information to the clinic is vital for effective development of the lead antibody.

Levels of rheumatoid factor isotypes, metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 in synovial fluid from various arthritides.

When synovial effusion is the only symptom, it is often difficult to make an exact diagnosis of the arthritic disease. To distinguish various types of arthritis with synovial effusion, we measured the levels of matrix metalloproteinase-3 (MMP-3, Stromelysin), tissue inhibitor of metalloproteinase-1 (TIMP-1) and rheumatoid factor (RF) isotypes in synovial fluid (SF) from patients with rheumatoid arthritis (RA), osteoarthritis (OA), pyogenic arthritis (PA), pseudogouty arthritis (PG), gouty arthritis (GA) and traumatic arthritis (TA). SF was aspirated from the knee joint or the ankle joint. Levels of IgG-, IgM- and IgA-RF isotypes were measured by ELISA. Levels of MMP-3 and TIMP-1 in SF were simultaneously determined by a one-step EIA system. Levels of IgG-RF, IgM-RF and MMP-3 in SF from RA patients were significantly higher than those in OA, PA, PG, GA and TA. However, IgA-RF in SF from RA patients, when compared with PA and GA, did not show a significantly increased level. In addition, TIMP-1 in SF from RA, when compared with PA and TA, also has not shown a significantly increased level. Therefore, in addition to analysing clinical data, measurements of IgG-RF, IgM-RF and MMP-3 in SF may contribute in distinguishing RA from other arthritic diseases.

Functional variation in endogenous and exogenous immunoglobulin binding to bovine neutrophils relative to parturition.

Ten healthy first- and second-lactation Holstein cows were observed from 1 week before to 1 week after calving and at postpartum day 30 to determine polymorphonuclear neutrophil (PMN) functional variation and immunoglobulin binding profiles. Blood and mammary PMN were obtained 3 times weekly and within 24 hours of calving. Functional traits measured included phagocytosis of Staphylococcus aureus and in vitro chemotaxis through micropore filters in a Boyden chamber. Additionally, PMN were evaluated for endogenous binding of IgG1, IgG2, IgA, and IgM before and after in vitro chemotaxis. Exogenous binding of the same isotypes was determined after incubation in pooled colostrum, purified immunoglobulin, and pooled sera. Phagocytosis results indicated a significant and transient increase in percentage of milk PMN with associated, rather than phagocytosed, bacteria for 1 week after calving. Blood PMN phagocytosis was not significantly different during this period. Though total chemotaxis was essentially unchanged, the percentage of PMN that were unable to complete migration increased substantially on the day of calving, an effect that disappeared by postpartum day 4. A significant (P < 0.01) positive correlation (r = 0.29) between percentage of PMN migrating completely through the micropore filter and percentage of blood PMN with associated bacteria was observed. Changes were not observed in endogenous immunoglobulin binding, with the exception of a peak in relative fluorescence intensity for IgG1 on the day of calving; this disappeared within 2 days after calving. Correlations between relative intensities of IgG2 and IgM, and percentage of mammary neutrophils phagocytosing were 0.37 and 0.70.(ABSTRACT TRUNCATED AT 250 WORDS)

Preferential mammary storage and secretion of immunoglobulin gamma (IgG) subclasses in swine.

Levels of three immunoglobulin gamma (IgG) subclasses, IgGA, IgGB and IgGC, were measured in sow sera, mammary glands, colostrum and milk samples by the single radial immunodiffusion. Serum IgGA and IgGB levels, but not IgGC, showed time dependent variations during gestation and lactation periods. The IgGA level started to decline at day 106 of gestation, reached its minimum at farrowing, and returned to the pre-gestation level 1-3 weeks after weaning. The serum IgGB level started to decrease at day 111 of gestation, reached its minimum at farrowing, and returned to the initial gestation level 1 week after farrowing. A notable decrease (P less than 0.1) in serum IgGC level was observed only on the day of farrowing. IgGA and IgGB were preferentially stored in mammary glands of full-term pregnant sows and secreted into colostrum after farrowing. In contrast, relatively small amounts of IgGC were stored in the mammary glands and secreted into colostrum. These data are interpreted as an indication that the preferential storage of IgGA and IgGB in the mammary gland of sows occurs at the time of significant decreases of these two IgG subclasses in the sera during late gestation and early lactation.

Characterization of the immunodeficiency of RIIS/J [corrected] mice. I. Association with the CD5 (LY-1) [corrected] B cell lineage.

RIIIS/J mice produce low antibody responses to several polysaccharide Ag of bacterial origin. They have low levels of serum IgM and IgG3 and high levels of serum IgG2a and IgG2b. Low serum IgM and IgG3 have been attributed to a low frequency of CD5 (Ly-1) B cells, which play an important role in the production of natural antibodies. Indeed, RIIIS/J mice have a low frequency of CD5 (Ly-1)+, IgM bright+, Ly-5 (B220)dull+ (i.e., CD5 (Ly-1) B) cells in their peritoneum. RIIIS/J mice treated with LPS produce a low anti-bromelain-treated mouse RBC splenic plaque-forming cell response and a normal anti-mouse transferrin splenic PFC response. Those data are compatible with the fact that CD5 (Ly-1) B cells contain the precursors of B lymphocytes secreting anti-bromelain-treated mouse RBC antibody. However, they have a higher frequency of IgM bright+, Mac-1+ cells in their peritoneum. These cells represent the CD5 (Ly-1) "sister population" of CD5 (Ly-1) B cells described by others. This suggests that characteristics usually associated with the CD5 (Ly-1) lineage are applicable only to the CD5 (Ly-1)+ Mac-1+ IgM+ population, but not the related CD5 (Ly-1)- Mac-1+ IgM+ population. RIIIS/J mice should thus prove a valuable model to study the CD5 (Ly-1) B cell lineage.

Unique V gene usage by B-Ly1 cell lines, and a discordance between isotype switch commitment and variable region hypermutation.

We have previously described a series of Ly-1+ B cell lines obtained as spontaneous outgrowth cultures from mouse spleen cells (B-Ly1 cells). In this study, we report the variable region sequences utilized by independent cell lines derived from three mouse strains. Remarkably, V region utilization and junctional diversification were essentially identical in all three cell lines. These included: lambda 1 light chain; JH1; DSP2-7,8; and, a novel VH gene segement. The VH was highly homologous to CP4, a rare VH family previously found in a group of Ly-1 B cell-derived autoantibodies to bromelain-treated mouse red blood cells. The selective expression of this unusual lg species implies the action of distinctive molecular or immunophysiological processes in the outgrowth of B-Ly1 cell lines. Clarification of these factors may be relevant to understanding the peculiar biology of Ly-1 B cells in vivo. We have also evaluated switch-committed B-Ly1 clones to determine whether this phenotype is accompanied by variable region somatic mutation. However, no evidence for somatic mutation was observed in these induced clones. This may indicate that these two processes are independently regulated, despite their common concordance in vivo.