Chronic immune complex disease in mice: the role of antibody affinity.

Two lines of mice selectively bred for producing high and low affinity antibody to protein antigens were repeatedly injected with human serum albumin and the severity and pattern of immune complex disease induced in this way was studied in the two lines. In low affinity mice, there was a greater intensity of deposits in the glomeruli shown by immunofluorescence, and more antibody was eluted from kidney homogenates compared to high affinity line mice. In the low affinity mice, complexes were mainly on the basement membrane whereas in high affinity mice, the localization of immune complexes was predominantly mesangial. However, no significant difference in glomerular filtration rates between the two lines was obtained. The immunopathological significance of antibody affinity is discussed in the light of these results.     

Direct binding of radioiodinated monoclonal antibody to tumor cells: significance of antibody purity and affinity for drug targeting or tumor imaging

For MoAb to be used efficiently for drug targeting and tumor imaging, the fraction of antibody binding to tumor cells must be maximized. We have studied the binding of 125I MoAb in three different tumor systems. The fraction of antibody that could be bound to the cell surface was directly proportional to the antibody purity. The affinity constant also limits the fraction of antibody that can bind to cells at a given antigen concentration. Rearrangement of the standard expression for univalent equilibrium binding between two reactants shows that in antigen excess, the maximum fraction of antibody that can bind (formula; see text). Binding data using four different MoAb with three cell systems confirm this relationship. Estimates for reasonable concentrations of tumor antigens in vivo indicate that antibodies with binding constants less than 10(8) M-1 are not likely to be useful for drug targeting or tumor imaging.     

Differences in immune elimination in inbred mice: the role of low affinity antibody

The rate of elimination of soluble protein antigen injected intravenously differs in different inbred strains of mice previously immunized to the antigen. This difference is apparently related to the affinity of the antibody they produce. Animals producing high affinity antibody eliminate antigen more effectively than those producing low affinity antibody. Passive transfer of antibody shows that the difference of antigen elimination is a property of the antibody, rather than the cellular mechanisms involved. The immunopathological significance of these findings is discussed.     

Cellular immunology tests applied to the diagnosis of immune system dysfunctions

In clinical immunology, improved standardisation and simplification have made skin and laboratory tests more amenable both to routine use and more rigorous interpretation. An individual's immune status can be assessed by the in vivo investigation of delayed hypersensitivity to widely distributed antigens or by techniques such as the mitogen test ot lymphoblast transformation, lymphocyte population and sub-population (e.g. T4, T8...) counts. A more delicate test of phagocytosis is provided by the chemoluminescence test. Investigation of allergic states (immediate hypersensitivity) relies on skin tests and also on laboratory techniques such as the basophil degranulation test.     

In vivo suppressive function of myeloid-derived suppressor cells is limited to the inflammatory site

Current paradigms suggest that, despite the heterogeneity of myeloid-derived suppressor cells (MDSC), all Gr-1(+) CD11b(+) cells can exert suppressive function when exposed to inflammatory stimuli. In vitro evaluation shows that MDSC from multiple tissue sites have suppressive activity, and in vivo inhibition of MDSC enhances T-cell function; however, the relative capacity of MDSC present at localized inflammatory sites or in peripheral tissues to suppress T-cell responses in vivo has not been directly evaluated. In the current study, we observed that during a tissue-specific inflammatory response, MDSC inhibition of CD8(+) T-cell proliferation and IFN-γ production was restricted to the inflammatory site. Using a prostate-specific inflammatory model and a heterotopic prostate tumor model, we showed that MDSC from inflammatory sites or from tumor tissue possess immediate capacity to inhibit T-cell function, whereas those isolated from peripheral tissues (spleens and liver) were not suppressive without activation of iNOS by exposure to IFN-γ. These data suggest that MDSC are important regulators of immune responses in the prostate during acute inflammation and the chronic inflammatory setting of tumor growth, and that regulation of T-cell function by MDSC during a localized inflammatory response is restricted in vivo to the site of an ongoing immune response.     

Adoptive T cell therapy: Boosting the immune system to fight cancer

Cellular therapies have shown increasing promise as a cancer treatment. Encouraging results against hematologic malignancies are paving the way to move into solid tumors. In this review, we will focus on T-cell therapies starting from tumor infiltrating lymphocytes (TILs) to optimized T-cell receptor-modified (TCR) cells and chimeric antigen receptor-modified T cells (CAR-Ts). We will discuss the positive preclinical and clinical findings of these approaches, along with some of the persisting barriers that need to be overcome to improve outcomes.     

In this issue--engineering the immune system to fight cancer and infections

As the immune system essentially evolved to fight off or keep at bay life-threatening infectious agents rather than cancer, the question remains as to how to best redeploy it for the treatment of a broader range of diseases. This is reflected by an unprecedented diversification of platform technologies in development, facilitated by rapid progress in biotechnology. In this issue, we host several contributions outlining major efforts in developing novel immune interventions spanning antigen-specific vaccination, non-antigen-targeted immune intervention, genetically engineered lymphocytes, and ultraspecific antigen-targeted ligands. In addition, the journal is hosting in this issue, two reviews discussing the complex matter and dynamic balance between immunity and viral infections, as the concept of fine modulation of that balance still carries the promise of yielding novel therapies.     

Relationship between antibody affinity and hemolytic plaque diameter—II: Maturation of primary immune responses to DNP and SRBC

The primary immune responses of C3H mice to the model monodeterminant antigen DNP2 and the multideterminant antigen SRBC were studied in order to determine the relationship between relative antibody affinity and the hemolytic plaque diameter of individual antibody-secreting cells during maturation of the primary immune responses. Competitive inhibition PFC assays, employing either DNP-lysine or SRBC stromata, were used to assess relative antibody affinity and the degree of heterogeneity. Both immune responses exhibited typical maturational features with a progressive increase in mean antibody affinity and a progressive decrease in the heterogeneity of antibody affinities. The mean diameter of anti-DNP plaques increased during maturation and high affinity anti-DNP antibody produced larger plaques than low affinity antibody. However, the mean plaque diamter of anti SRBC plaques decreased during maturation. This discrepancy was explored by analysis of the distribution of PFC sizes and the capacity of large vs small plaques to be neutralized by competing antigen. The anti-SRBC response was characterized by a significantly greater frequency of small plaques during maturation as compared to the anti-DNP response. These new small plaques were less susceptible to inhibition than the larger plaques, a result consistent with the progressive emergence of PFC producing low affinity antibody. This study suggests that immunization with a complex multideterminant antigen such as SRBC may significantly influence the distribution of plaque sizes. Whereas competitive inhibition PFC analyses may provide a relative measure of antibody affinity and the clonal distribution of antibody affinities in responses to both monodeterminant and multideterminant antigens, analysis of maturation by reference to plaque diameter per se wean be erroneous, owing perhaps to the appearance of subpopulations of PFC synthesizing low affinity antibody or antibody directed to less dense determinants.     

In vitro improvement of a shark IgNAR antibody by Qbeta replicase mutation and ribosome display mimics in vivo affinity maturation

We have employed a novel mutagenesis system, which utilizes an error-prone RNA dependent RNA polymerase from Qbeta bacteriophage, to create a diverse library of single domain antibody fragments based on the shark IgNAR antibody isotype. Coupling of these randomly mutated mRNA templates directly to the translating ribosome allowed in vitro selection of affinity matured variants showing enhanced binding to target, the apical membrane antigen 1 (AMA1) from Plasmodium falciparum. One mutation mapping to the IgNAR CDR1 loop was not readily additive to other changes, a result explained by structural analysis of aromatic interactions linking the CDR1, CDR3, and Ig framework regions. This combination appeared also to be counter-selected in experiments, suggesting that in vitro affinity maturation is additionally capable of discriminating against incorrectly produced protein variants. Interestingly, a further mutation was directed to a position in the IgNAR heavy loop 4 which is also specifically targeted during the in vivo shark response to antigen, providing a correlation between natural processes and laboratory-based affinity maturation systems.     

In vivo and ex vivo magnetic resonance spectroscopy as applied to pharmacokinetic studies with anticancer agents: a review.

The potential role of MRS in studying the pharmacokinetics of anticancer drugs is reviewed. In vivo and ex vivo MRS has been used extensively in studies with fluoropyrimidines. Results from preclinical models have demonstrated that biochemical modulation of 5-fluorouracil metabolism can be demonstrated by MRS. The more general potential of MRS is illustrated by studies with the antifolate CB3988 (C2-desamino-C2-methyl-N10-propargyl-2'-trifluoromethyl-5,8-dideazafolic acid). Studies in mice and rats have shown that hepatobiliary clearance and renal elimination can be measured non-invasively by MRS. Comparison of half-lives derived from MRS and high performance liquid chromatography data gave reasonable agreement. In addition, MRI was used to localize drug-derived material within the abdominal cavity. The application of ex vivo MRS is illustrated by studies on the urinary excretion of platinum complexes. 1H-MRS has been used to demonstrate the presence of the cyclobutanedicarboxylate leaving group, both free and platinum bound, in the urine of patients treated with carboplatin. With iproplatin 195Pt NMR has been used to demonstrate in vivo reduction of this Pt(IV) complex to Pt(II) complexes. Finally, the application of MRS to the study of the molecular pharmacology of alkylating agents (a nitrogen mustard and N-methyl-N-nitrosourea) is discussed.     

Studies on the control of antibody synthesis: III. Changes in heterogeneity of antibody affinity during the course of the immune response

The distribution and heterogeneity of antibody affinity has been followed with time after immunization. Initially, a symmetrical distribution of heterogeneous low affinity antibody molecules is present. With time the population becomes skewed towards the high affinity end of the distribution, the average affinity increases, and the bulk of the antibody generally comes to be present in a subpopulation of high affinity and of relatively restricted heterogeneity. Still later after immunization, the proportion of high affinity antibody decreases and a highly heterogeneous population of antibody molecules is present with a somewhat decreased average affinity. Low affinity subpopulations were found to persist throughout the course of the immune response. In addition it was noted that by day 42 after immunization a significant amount of the highest affinity antibody that a given rabbit would synthesize at any time during its immune response was already present. Thus, changes in average affinity can be accounted for by shifts in the relative amounts of those antibody subpopulations already present by day 42 and do not require the appearance of any new antibody species. The results can be interpreted as consistent with a selection theory of antibody synthesis in which cells of high affinity are preferentially selected in what amounts to a micro-evolutionary system.     

T-cell-dependent oscillations of IgM antibody affinity during the immune response to DNP-Dextran to low or high epitope density.

Anti-hapten IgM antibody response and affinity were evaluated by haemolytic plaque inhibition assay on spleen cells from mice immunized with 2,4, dinitrophenyl (DNP)1.3-Dextran or DNP13-Dextran. Regardless of epitope density, affinity was found to mature with time after immunization and to be characterized by rapid oscillations independent of changes in anti-hapten plaque-forming cell (PFC) response and antibody secretion rate. Injection of the lower epitope density immunogen not only elicited higher PFC responses and higher affinity antibodies but also induced more pronounced affinity oscillations mainly confined to the higher affinity PFC subpopulation. Immunization of athymic nude mice with DNP1.3-Dextran elicited PFC responses comparable to those observed in similarly immunized euthymic mice. However, affinity oscillations were drastically reduced in athymic mice and the restricted variation of antibody affinity observed shortly after immunization was followed by no oscillations in any of the affinity PFC subpopulations. Athymic mice did not produce high affinity PFC which account to a large extent for the affinity oscillations observed in euthymic mice. These findings demonstrate the important role of T cells in the appearance of rapid changes in IgM antibody affinity. The generation of T-cell-dependent oscillations of high affinity antibody-producing cell subpopulations is discussed in terms of interactions among cells and soluble factors involved in regulatory circuits of the immune network.     

In vivo and in vitro genetic evidence of involvement of neuregulin 1 in immune system dysregulation

Neuregulin 1 (NRG1) has been implicated in several disorders including breast cancer, multiple sclerosis, and schizophrenia. Also, recent evidence suggests that NRG1 may play a role in regulation of inflammation and immune system response. We therefore hypothesized that a schizophrenia-associated missense mutation (valine to leucine) we identified within the transmembrane region of NRG1 would also be linked to immune dysregulation. We used plasma samples from families carrying the mutation to measure levels of antibodies to 41 autoimmune markers and six cytokines (IL-1b, IL-6, IL-10, IL-8, IL-12p70, and TNF-α) and used these levels as quantitative traits to evaluate association with the NRG1 mutation, using FBAT. Next, we used Epstein–Barr virus-transformed B cells from heterozygous mutation carriers and wild-type individuals to evaluate protein and mRNA cytokine expression in vitro using quantitative PCR and ELISA assays. In vivo, increased levels of 25 autoimmune markers as well as elevated levels of cytokines were significantly associated with the NRG1 mutation. In vitro, we observed a significant increase in protein secretion levels of IL-6, TNF-α, and IL-8 in mutation carriers compared with controls. At the mRNA level, we observed a significant increase in IL-6 expression, while IL-4 levels appeared to be down-regulated in heterozygous individuals compared with wild-type controls. This is the first report of association of a NRG1 mutation with immune dysregulation. This study could contribute towards understanding the role of NRG1 in the pathogenesis of schizophrenia and other disorders in which inflammation plays an important role.     

A neutralizing antibody to the a chain of abrin inhibits abrin toxicity both in vitro and in vivo

Plant ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that inhibit protein synthesis in cells. Abrin, a type II RIP, is an AB type toxin, which is one of the most lethal types of toxin known. The B chain facilitates the entry of the molecule into the cell, whereas the A chain exerts the toxic effect. We have generated hybridomas secreting antibodies of the immunoglobulin G class specific to the recombinant A chain of abrin. One monoclonal antibody, namely, D6F10, rescued cells from abrin toxicity. Importantly, the antibody also protected mice from lethal doses of the toxin. The neutralizing effect of the antibody was shown to be due to interference with abrin attachment to the cell surface.     

HBsAg: anti-HBs immune complexes. A method for separating the constituent components and assessment of the affinity of the antibody

A number of chemical disruption agents were assessed for their ability to dissociate HBsAg:anti-HBs immune complexes and to release both the antibody and antigen component in immunologically active forms. The most appropriate reagent was 0.1 M diethylamine which could elute up to 81% of anti-HBs antibody bound to solid-phase HBsAg and retained 93% of its antigen-combining activity. Complexes formed at various degrees of antigen excess and pre-exposed to 0.1 M diethylamine at room temperature for 18 h before ultracentrifugation on sucrose density gradients were effectively dissociated. The released antibody and antigen banded at their expected densities. However, the affinity of the isolated antibody for the detergent-solubilized polypeptide complex from purified HBsAg (gp30/p25) and cyclical peptides representing amino acids 124-137 and 139-147 of HBsAg were found to be considerably lower than that of the original pooled anti-HBs immunoglobulin used to form the immune complexes. These results suggest that the highest affinity antibody subpopulation may not be completely dissociated from the complex. Care should thus be exercised in the interpretation of the significance of the observed affinity of the antibody isolated by this and other similar dissociation procedures.