Marvin B. Rittenberg

Enhancement and destruction of antibody function by somatic mutation: unequal occurrence is controlled by V gene combinatorial associations.

We examined the positive and negative effects of somatic mutation on antibody function using saturation mutagenesis in vitro to mimic the potential of the in vivo process to diversify antibodies. Identical mutations were introduced into the second complementarity determining region of two anti‐phosphocholine antibodies, T15 and D16, which share the same germline VH gene sequence. T15 predominates in primary responses and does not undergo affinity maturation. D16 is representative of antibodies that co‐dominate in memory responses and do undergo affinity maturation. We previously reported that > 50% of T15 mutants had decreased antigen binding capacity. To test if this high frequency of binding loss was unique to T15 or a consequence of random point mutations applicable to other combining sites, we analyzed the same mutations in D16. We show that D16 suffers a similar loss of function, indicating an equally high potential for B‐cell wastage. However, only D16 displayed the capacity for somatic mutation to improve antigen binding, which should enhance its persistence in memory responses. Mutation of residues contacting the haptenic group, as determined by molecular modeling, did not improve binding. Instead, productive mutations occurred in residues that either contacted carrier protein or were distant from the antigen binding site, possibly increasing binding site flexibility through long‐range effects. Targeting such residues for mutation should aid in the rational design of improved antibodies.

The Adaptive Potential of the Memory Response: Clonal Recruitment and Epitope Recognition

The expression of immune memory represents the culmination of the immune systems ability to respond to antigen. In the absence of memory, the response is relatively slow and limited in scope. Maturation to memory alters this pattern; substantial changes in antibodies occur between the primary and the memory response that affect the recognition of antigen. These changes reflect diversification of the antibody combining sites participating in the response and enhance their ability to deal with a renewed antigenic challenge from the infectious agent. The recent application of molecular genetics to studies of immune memory made it clear that both somatic mutation and recruitment of new clonotypes contribute to the molecular diversity of the memory response {see volume 96 of the journal for recent reviews). The temporal progression in antibody diversity that results from these mechanisms is an adaptive response that appears to follow a Darwinian process in which limiting amounts of antigen are able to provide the selective pressure for the structures of best fit, i.e. surface immunoglobulin. Thus, memory development and expression is a dynamic process in which epitope recognition may vary depending on the composition of the antibody population at any particular moment. We have found the memory response to phosphocholine (PC) protein conjugates to consist of two competing antibody populations with distinct antigen-binding phenotypes. One of these populations predominates in the primary response while the second appears late hut achieves codominance

Harmful somatic mutations: Lessons from the dark side

Summary: The ability of somatic mutation to modify the course of an immune response is well documented. However, emphasis has been placed almost exclusively on the ability of somatic mutation to improve the functional characteristics of representative antibodies. The harmful effects of somatic mutation, its dark side, have been far less well characterized. Yet evidence suggests that the number of B cells directed to wastage pathways as a result of harmful somatic mutation probably far exceeds the number of cells whose antibodies have been improved. Here we review our recent findings in understanding the structural and functional consequences of V‐region mutation.

ENDOTOXIN REGULATES CORTICOTROPIN-RELEASING HORMONE RECEPTOR 2 IN HEART AND SKELETAL MUSCLE

We tested the effect of endotoxin on the peripheral corticotropin-releasing hormone receptor (CRH-R2), which is highly expressed in the heart. Systemic injection of LPS markedly downregulated CRH-R2 mRNA levels in the heart in a dose and time dependent manner. In contrast, CRH-R2 levels in skeletal muscle increased following exposure to endotoxin. These results suggest that CRH-R2 may be differentially regulated in cardiac tissue and skeletal muscle. Finding that CRH-R2 expression in the heart is modulated by endotoxin, a potent inducer of cardiovascular dysregulation, suggests a possible link between CRH and the cardiovascular response to stress.

Mutation of a Single Conserved Residue in VH Complementarity-Determining Region 2 Results in a Severe Ig Secretion Defect

During an immune response, somatic mutations are introduced into the VH and VL regions of Ig chains. The consequences of somatic mutation in highly conserved residues are poorly understood. Ile51 is present in 91% of murine VH complementarity-determining region 2 sequences, and we demonstrate that single Ile51→Arg or Lys substitutions in the PCG1-1 Ab are sufficient to severely reduce Ig secretion (1–3% of wild-type (WT) levels). Mutant H chains, expressed in the presence of excess L chain, associate with Ig binding protein (BiP) and GRP94 and fail to form HL and H2L assembly intermediates efficiently. The mutations do not irreversibly alter the VH domain as the small amount of mutant H chain, which assembles with L chain as H2L2, is secreted. The secreted mutant Ab binds phosphocholine-protein with avidity identical with that of WT Ab, suggesting that the combining site adopts a WT conformation. A computer-generated model of the PCG1-1 variable region fragment of Ig (Fv) indicates that Ile51 is buried between complementarity-determining region 2 and framework 3 and does not directly contact the L chain. Thus, the Ile51→Arg or Ile51→Lys mutations impair association with the PCG1-1 L chain via indirect interactions. These interactions are in part dependent on the nature of the L chain as the PCG1-1 VH single Ile51→Arg or Ile51→Lys mutants were partially rescued when expressed with the J558L λ1 L chain. These results represent the first demonstration that single somatic mutations in VH residues can impair Ig secretion and suggest one reason for the conservation of Ile51 in so many Ig VH.

A Genetic Model of Stress Displays Decreased Lymphocytes and Impaired Antibody Responses Without Altered Susceptibility to Streptococcus pneumoniae

Stress pathways affect immune function, the most notable of these pathways being activation of the hypothalamic-pituitary-adrenal (HPA) axis. Although HPA activation has generally been relegated to an immunosuppressive role, recent evidence suggests that stress and HPA activation can be immunoenhancing in certain situations. To investigate specific effects of stress on immune function, we used a genetic model of chronic stress wherein transgenic mice overexpress corticotropin-releasing hormone (CRH), a primary mediator of the stress response. In these mice, CRH is overproduced in the brain, leading to chronic activation of the HPA axis. We found that CRH-transgenic mice have decreased leukocyte numbers in lymphoid compartments, with preferential loss of B lymphocytes. They also exhibit decreased Ab production and impaired isotype switching in response to immunization with a thymus-dependent Ag, phosphocholine-keyhole limpet hemocyanin. Despite these deficits, immunization protected CRH-transgenic and wild-type mice equally well against lethal challenge with Streptococcus pneumoniae, an encapsulated Gram-positive bacterium known to require Ab-mediated opsonization for clearance. While IgG responses are severely depressed in these mice, IgM titers are only modestly decreased. This fairly robust IgM response may be sufficient to protect against S. pneumoniae. Additionally, while total leukocyte numbers are decreased in these mice, neutrophil numbers are increased. This increase in number of neutrophils may compensate for the depressed IgG response, allowing adequate host defense during chronic stress.

Combining site specificity of monoclonal antibodies to the organophosphate hapten soman

The combining site specificities of eight monoclonal antibodies raised against the organophosphorous-containing hapten Soman are compared to monoclonal antibodies specific for a naturally occurring organophosphorous compound, phosphocholine (PC). Although these haptens share some structural and spatial features, differences in their chemical structures, most notably the presence or absence of a positive charge, appear to prevent significant cross-reactivity between antibodies specific for each. The murine memory response to PC-KLH has been shown previously to be characterized by the presence of two major groups of antibodies differentiated on the basis of their specificity for free PC and for the nitrophenyl derivative of PC, nitrophenylphosphocholine (NPPC). Interestingly, two groups of hybridoma antibodies were detected in the immune response to Soma-KLH which possess differential specificity for Soman and for a nitrophenyl derivative of Soman.

Antibody combining site heterogeneity within the response to phosphocholine-keyhole limpet hemocyanin

The memory response to PC-KLH is dominated by two antibody populations differing in fine specificity. Group I antibodies show affinity for both phosphocholine (PC) and p-nitrophenyl phosphocholine (NPPC). Group II antibodies exhibit significant affinity only for NPPC. Here, we describe the binding site characteristics of Group II antibodies and show that in recognizing NPPC these antibodies have a common requirement for the phenyl moiety, a negatively charged phosphate, and the trimethyl structure of the choline. However, Group II antibodies were found to differ in their requirement for the positively charged nitrogen of choline and thus could be divided into two subgroups. In contrast to Group II-A, Group II-B antibodies recognize not only NPPC but also its analog p-nitrophenyl-3,3-dimethyl butyl phosphate (NPDBP), which differs from NPPC by substituting a carbon for the positively charged nitrogen of the choline moiety. These results suggest that Group II-B antibodies do not require the positive charge in order to bind, although the binding constant, Ka, was increased when the nitrogen was present. Furthermore, heterogeneity within Group II antibodies was characterized by differences in binding to dinitrophenyl phosphocholine which has an additional phenyl ring and aminophenyl phosphocholine which has an amino group in place of the nitro group of NPPC. The results indicate that diversity in the memory response to PC-KLH is reflected in the Group II antigen-binding phenotype by antibodies which differ appreciably in their recognition of various structural aspects of the hapten.

Replacements in the exposed loop of the T15 antibody VH CDR2 affect carrier recognition of PC-containing pathogens

A panel of mutant antibodies of the phosphocholine (PC)-binding antibody, T15, was tested for binding to PC-protein, Streptococcus pneumoniae, Trichinella spiralis and Ascaris suum. Relative to wildtype T15, all the mutant antibodies showed differential recognition of the panel of PC-associated antigens. These mutant antibodies contain amino acid replacements in the CDR2 region of the heavy chain variable region, indicating the importance of CDR2 in recognition of carrier determinants. A model of T15 is shown that illustrates the strategic placement of mutations that could allow interaction with determinants associated with PC. A direct implication of this finding is that the T15 antibody combining site accommodates structures larger than phosphocholine and that recognition of associated carrier determinants could be a significant force in shaping the immune response to PC-containing pathogens.

Repertoire Shift in the Humoral Response to Phosphocholine-Keyhole Limpet Hemocyanin: VH Somatic Mutation in Germinal Center B Cells Impairs T15 Ig Function

Phosphocholine (PC) is a naturally occurring Ag common to many pathogenic microorganisms. Early in the primary response to PC conjugated to keyhole limpet hemocyanin (KLH), T15 Id+ Abs constitute >90% of the serum Ig in BALB/c mice. During the late primary and memory response to PC-protein, a shift in the repertoire occurs and T15 Id+ Abs lose dominance. In this study, we use immunohistochemistry and single germinal center microdissection to locate T15 Id+ cells in the spleen in a primary response to PC-KLH. We demonstrate T15 Id+ B cells and VH1-DFL16.1-JH1 and Vκ22-Jκ5 rearrangements in germinal centers early in the immune response; thus loss of T15 dominance is not due to lack of T15 cells within germinal centers. One-hundred thirty one VH1 and 57 Vκ22 rearrangements were cloned and sequenced. Thirty four percent of the VH1 clones and 37% of the Vκ22 clones contained somatic mutations indicating participation in the germinal center response. Six variant T15 H clones were expressed with wild-type T15 L chain in vitro. Two of these Abs were defective in secretion providing the first evidence that mutation occurring in vivo can disrupt Ig assembly and secretion. Of the four secretion-competent Abs, two failed to display binding to PC-protein, while the other two displayed altered carrier recognition. These results indicate that somatic mutation of T15 in vivo can result in the loss of binding and secretion, potentially leading to B cell wastage. The failure of T15 to gain affinity enhancing mutations in the face of these detrimental changes may contribute to repertoire shift.