The MutSß complex is a modulator of p53-driven tumorigenesis through its functions in both DNA double-strand break repair and mismatch repair.

Loss of the DNA mismatch repair (MMR) protein MSH3 leads to the development of a variety of tumors in mice without significantly affecting survival rates, suggesting a modulating role for the MutSß (MSH2-MSH3) complex in late-onset tumorigenesis. To better study the role of MSH3 in tumor progression, we crossed Msh3(-/-) mice onto a tumor predisposing p53-deficient background. Survival of Msh3/p53 mice was not reduced compared with p53 single mutant mice; however, the tumor spectrum changed significantly from lymphoma to sarcoma, indicating MSH3 as a potent modulator of p53-driven tumorigenesis. Interestingly, Msh3(-/-) mouse embryonic fibroblasts displayed increased chromatid breaks and persistence of γH2AX foci following ionizing radiation, indicating a defect in DNA double-strand break repair (DSBR). Msh3/p53 tumors showed increased loss of heterozygosity, elevated genome-wide copy-number variation and a moderate microsatellite instability phenotype compared with Msh2/p53 tumors, revealing that MSH2-MSH3 suppresses tumorigenesis by maintaining chromosomal stability. Our results show that the MSH2-MSH3 complex is important for the suppression of late-onset tumors due to its roles in DNA DSBR as well as in DNA MMR. Further, they demonstrate that MSH2-MSH3 suppresses chromosomal instability and modulates the tumor spectrum in p53-deficient tumorigenesis and possibly has a role in other chromosomally unstable tumors as well.

Both Th1 and Th2 cytokines affect the ability of monoclonal antibodies to protect mice against Cryptococcus neoformans.

Variable-region-identical mouse immunoglobulin G1 (IgG1), IgG2b, and IgG2a monoclonal antibodies to the capsular polysaccharide of Cryptococcus neoformans prolong the lives of mice infected with this fungus, while IgG3 is either not protective or enhances infection. CD4+ T cells are required for IgG1-mediated protection, and CD8+ T cells are required for IgG3-mediated enhancement. Gamma interferon is required for both effects. These findings revealed that T cells and cytokines play a role in the modulation of cryptococcal infection by antibodies and suggested that it was important to more fully define the cytokine requirements of each of the antibody isotypes. We therefore investigated the efficacy of passively administered variable-region-identical IgG1, IgG2a, IgG2b, and IgG3 monoclonal antibodies against intravenous infection with C. neoformans in mice genetically deficient in interleukin-12 (IL-12), IL-6, IL-4, or IL-10, as well as in the parental C57BL/6J strain. The relative inherent susceptibilities of these mouse strains to C. neoformans were as follows: IL-12(-/-) > IL-6(-/-) > C57BL/6J approximately IL-4(-/-) >> IL-10(-/-). This is consistent with the notion that a Th1 response is necessary for natural immunity against cryptococcal infection. However, none of the IgG isotypes prolonged survival in IL-12(-/-), IL-6(-/-), or IL-4(-/-) mice, and all isotypes significantly enhanced infection in IL-10(-/-) mice. These results indicate that passive antibody-mediated protection against C. neoformans requires both Th1- and Th2-associated cytokines and reveal the complexity of the mechanisms through which antibodies modulate infection with this organism.

Testing the reverse transcriptase model of somatic mutation.

Somatic hypermutation of the variable (V) regions of rearranged immunoglobulin genes leads to antibody affinity maturation. Although this process has been extensively studied, the mechanisms responsible for these multiple point mutations are still elusive. One mechanism that was proposed over 10 years ago by Steele and Pollard was that an intrinsic reverse transcriptase (RT) copies the nascent mRNA creating the large number of observed point mutations due to its high error rate. A cDNA copy of the mutated V region would then replace the endogenous DNA through a gene conversion-like event, thus integrating these point mutations into the genome. This model of hypermutation would account for the very high mutation rate, the presence of hotspots, strand bias, the requirement for transcription and localization of mutation within the immunoglobulin V region. Using AZT and ddC to inhibit endogenous RTs, we have assayed for somatic mutation using a murine in vivo model. Somatic mutation occurred at similar frequencies and with the same characteristics with or without treatment of RT inhibitors, suggesting that standard reverse transcription is not required for antibody V region hypermutation in the mouse.

Clonal instability of V region hypermutation in the Ramos Burkitt's lymphoma cell line.

Affinity maturation of the humoral immune response is caused by single base changes that are introduced into the V regions of the Ig genes during a brief period of B cell differentiation. It has recently become possible to study V region mutation in some human Burkitt's lymphoma cell lines that mutate their V regions and express surface markers that suggest they arose from the malignant transformation of germinal center B cells. Ramos Burkitt's cells constitutively mutate their V regions at a rate of approximately 2 x 10(-5) mutations/bp/generation. However, the sequencing of unselected V regions suggested that our Ramos cell line was progressively losing its ability to undergo V region hypermutation. To accurately quantify this process, subclones with different nonsense mutations in the mu heavy chain V region were identified. Reversion analysis and sequencing of unselected V regions were used to examine the clonal stability of V region hypermutation. Even after only 1 month in culture, stable and unstable subclones could be identified. The identification of mutating and non-mutating subclones of Ramos provided a unique opportunity to identify factors involved in the mutational process. Differential gene expression between mutating and non-mutating Ramos clones was examined by RT-PCR and cDNA microarray analyses. We found that the expression of activation-induced cytidine deaminase (AID), a putative cytidine deaminase, correlated with mutation rates in Ramos subclones. These results suggest that the hypermutation phenotype is inherently unstable in Ramos and that long culture periods favor outgrowth of non-mutating cells that express lower levels of AID.

Expression of error-prone polymerases in BL2 cells activated for Ig somatic hypermutation.

High affinity antibodies are generated in mice and humans by means of somatic hypermutation (SHM) of variable (V) regions of Ig genes. Mutations with rates of 10(-5)--10(-3) per base pair per generation, about 10(6)-fold above normal, are targeted primarily at V-region hot spots by unknown mechanisms. We have measured mRNA expression of DNA polymerases iota, eta, and zeta by using cultured Burkitt's lymphoma (BL)2 cells. These cells exhibit 5-10-fold increases in heavy-chain V-region mutations targeted only predominantly to RGYW (R = A or G, Y = C or T, W = T or A) hot spots if costimulated with T cells and IgM crosslinking, the presumed in vivo requirements for SHM. An approximately 4-fold increase pol iota mRNA occurs within 12 h when cocultured with T cells and surface IgM crosslinking. Induction of pols eta and zeta occur with T cells, IgM crosslinking, or both stimuli. The fidelity of pol iota was measured at RGYW hot- and non-hot-spot sequences situated at nicks, gaps, and double-strand breaks. Pol iota formed T x G mispairs at a frequency of 10(-2), consistent with SHM-generated C to T transitions, with a 3-fold increased error rate in hot- vs. non-hot-spot sequences for the single-nucleotide overhang. The T cell and IgM crosslinking-dependent induction of pol iota at 12 h may indicate an SHM "triggering" event has occurred. However, pols iota, eta, and zeta are present under all conditions, suggesting that their presence is not sufficient to generate mutations because both T cell and IgM stimuli are required for SHM induction.

Somatic hypermutation in MutS homologue (MSH)3-, MSH6-, and MSH3/MSH6-deficient mice reveals a role for the MSH2-MSH6 heterodimer in modulating the base substitution pattern.

Although the primary function of the DNA mismatch repair (MMR) system is to identify and correct base mismatches that have been erroneously introduced during DNA replication, recent studies have further implicated several MMR components in somatic hypermutation of immunoglobulin (Ig) genes. We studied the immune response in mice deficient in MutS homologue (MSH)3 and MSH6, two mutually exclusive partners of MSH2 that have not been examined previously for their role in Ig hypermutation. In Msh6(-)/- and Msh3(-)/-/Msh6(-)/- mice, base substitutions are preferentially targeted to G and C nucleotides and to an RGYW hot spot, as has been shown previously in Msh2(-)/- mice. In contrast, Msh3(-)/- mice show no differences from their littermate controls. These findings indicate that the MSH2-MSH6 heterodimer, but not the MSH2-MSH3 complex, is responsible for modulating Ig hypermutation.

Influence of affinity and antigen density on antibody localization in a modifiable tumor targeting model.

A persistent question in the field of antibody imaging and therapy is whether increased affinity is advantageous for the targeting of tumors. We have addressed this issue by using a manipulatable model system to investigate the impact of affinity and antigen density on antibody localization. In vitro enzyme-linked immunosorbent assays and bead-binding assays were carried out using BSA conjugated with high and low densities (HD and LD, respectively) of the chemical hapten rho-azophenyl-arsonate as an antigen. Isotype-matched monoclonal antibodies (mAbs) 36-65 and 36-71, with identical epitope specificity but 200-fold differences in affinity, were chosen as targeting agents. The relative in vitro binding of 36-65 and 36-71 was compared with an artificial "tumor" model in vivo using antigen-substituted beads s.c. implanted into SCID mice. Nonsubstituted BSA beads were implanted in the contralateral groin as a nonspecific control. The efficacy of the targeting of [125I]-labeled antibodies was assessed by the imaging of animals on a gamma-scintillation camera using quantitative region-of-interest image analysis over the course of 2 weeks and by postmortem tissue counting. In vitro, both antibodies bound well to the HD antigen, whereas only the high-affinity mAb 36-71 bound effectively to the LD antigen. In vivo, high-affinity mAb 36-71 bound appreciably to both LD and HD beads. In contrast, there was no specific localization of low-affinity mAb 36-65 to LD antigen beads, although the antibody did bind to the beads with the HD antigen. Whereas the high-affinity mAb 36-71 increased its binding to HD beads throughout the 14 days of observation, binding of the high affinity antibody to LD beads and of the low affinity antibody to HD beads plateaued between 10-14 days. These in vitro and in vivo findings demonstrate that the need for a high-affinity antibody is dependent on the density of the target antigen. High-affinity antibodies bind effectively even with a single antigen-Fab interaction, irrespective of the antigen density. In contrast, low-affinity antibodies, because of weak individual antigen-Fab interactions, require the avidity conferred by divalent binding for effective attachment, which can only occur if antigen density is above a certain threshold. An understanding of the differential behavior of high- and low-affinity antibodies and the impact of avidity is useful in predicting the binding of monovalent antibody fragments and engineered antibody constructs and underlies the trend toward development of multivalent immunological moieties. Consideration of the relative density of the antigen on the tumor and the background tissues may enable and even favor targeting with low-affinity antibodies in selected situations.

Molecular and idiotypic analyses of the antibody response to Cryptococcus neoformans glucuronoxylomannan-protein conjugate vaccine in autoimmune and nonautoimmune mice.

The antibody response to Cryptococcus neoformans capsular glucuronoxylomannan (GXM) in BALB/c mice frequently expresses the 2H1 idiotype (Id) and is restricted in variable gene usage. This study examined the immunogenicity of GXM-protein conjugates, V (variable)-region usage, and 2H1 Id expression in seven mouse strains: BALB/c, C57BL/6, A/J, C3H, NZB, NZW, and (NZB x NZW)F(1) (NZB/W). All mouse strains responded to vaccination with GXM conjugated to tetanus toxoid (TT), the relative magnitude of the antibody response being BALB/c approximately C3H > C57BL/6 approximately NZB approximately NZW approximately NZB/W > A/J. Analysis of serum antibody responses to GXM with polyclonal and monoclonal antibodies to the 2H1 Id revealed significant inter- and intrastrain differences in idiotype expression. Thirteen monoclonal antibodies (MAbs) (two immunoglobulin M [IgM], three IgG3, one IgG1, three IgG2a, two IgG2b, and two IgA) to GXM were generated from one NZB/W mouse and one C3H/He mouse. The MAbs from the NZB/W mouse were all 2H1 Id positive (Id(+)) and structurally similar to those previously generated in BALB/c mice, including the usage of a V(H) from the 7183 family and Vkappa5.1. Administration of both 2H1 Id(+) and Id(-) MAbs from NZB/W and C3H/H3 mice prolonged survival in a mouse model of cryptococcosis. Our results demonstrate (i) that V-region restriction as indicated by the 2H1 Id is a feature of both primary and secondary responses of several mouse strains; and (ii) that there is conservation of V-region usage and length of the third complementarity-determining region in antibodies from three mouse strains. The results suggest that V-region restriction is a result of antibody structural requirements necessary for binding an immunodominant antigen in GXM.

Chimeric human-mouse IgG antibodies with shuffled constant region exons demonstrate that multiple domains contribute to in vivo half-life.

Structural features that determine the differing rates of immunoglobulin catabolism are of great relevance to the engineering of immunologically active reagents. Sequences in the CH2 and CH3 region of IgG have been shown to regulate the rate of clearance through their interaction with FcRn. In an attempt to probe additional structural features that regulate antibody half-life, we have investigated two families of chimeric antibodies, composed of identical murine heavy and light antidansyl variable regions joined to human kappa light-chains and wild-type or shuffled human IgG heavy-chain constant regions. These antibodies were iodinated, and their clearance was studied in severe combined immunodeficient mice hosts by whole-body radioactivity measurements. Clearances of the wild-type and recombinant antibodies were biphasic. In a panel of immunoglobulins derived from IgG2 and IgG3, as successive domains were varied from gamma2 to gamma3, beta-phase half-life gradually decreased from 337.0 h to 70.6 h. Statistical analysis suggested that the composition of each of the three domains affected half-life, and no single region of the molecule by itself determined the rate of clearance. In the second panel of immunoglobulins derived from IgG1 and IgG4, the construct with the amino terminus portion of the molecule derived from IgG4, joined within the CH2 domain to the COOH terminus portion of IgG1, had a half-life paradoxically greater than either IgG1, or IgG4 (P < 0.012). All four IgG1/IgG4 constructs demonstrated presence of the concentration catabolism phenomenon, which is a unique hallmark of immunoglobulin catabolism. The contribution of all three constant region domains to immunoglobulin half-life may be due to distant conformational effects in addition to direct binding to protective receptors, and emphasizes the importance of distant sequences on the rate of immunoglobulin catabolism. Interesting possibilities regarding mechanisms controlling immunoglobulin metabolism are raised by the hybrid gamma4/gamma1 molecule with a half-life greater than either parental immunoglobulin. Understanding the relationships between the structure of these molecules and their clearance rate will further our ability to produce immunoglobulins with improved pharmacokinetic properties.

The effects of E mu, 3'alpha (hs 1,2) and 3'kappa enhancers on mutation of an Ig-VDJ-Cgamma2a Ig heavy gene in cultured B cells.

The Ig kappa intronic and 3'kappa light chain enhancers have been shown to be necessary for V region hypermutation in kappa light chain transgenes. To investigate the role of the E mu intronic enhancer in V region hypermutation of heavy chain genes, E mu and its associated matrix attachment regions (MAR) were replaced with the SV40 or the cytomegalovirus (CMV) enhancer in a gamma2a construct that hypermutates its rearranged VDJ region in the NSO plasmacytoma and 18.81 pre-B cell lines. In this model in vitro system, mutation rates of stable transfectants were determined by reversion analysis using a V region stop codon that, when mutated, allowed the detection of cellular revertants by ELISA spot assay. The gamma2a constructs with the E mu, SV40 and CMV enhancers mutated at comparably high rates in the B cell lines, but not in L cells, indicating that the E mu enhancer and its associated MAR were not specifically required for IgH hypermutation in this system. In parallel experiments, the addition of the 3'alpha heavy chain enhancer (hs 1,2) or the 3'kappa light chain enhancer did not increase the mutation rate of a related mu reporter construct in which the associated VDJ mutates at a moderately low rate in NSO cells or in cell hybrids made between 18.81 and NSO. These results imply that cis-acting IgH elements that promote hypermutation may not be restricted to Ig-specific transcriptional enhancers.

Aspects of antigen mimicry revealed by immunization with a peptide mimetic of Cryptococcus neoformans polysaccharide.

We have recently identified peptide mimetics of the Cryptococcus neoformans capsular polysaccharide by screening phage display peptide libraries. 2H1, one of a large family of mAbs against the glucuronoxylomannan fraction (GXM), is highly protective and binds several peptide motifs. This study analyzes the immunologic properties of P601E (SYSWMYE), a peptide from the low affinity motif (W/YXWM/LYE) that has an extended cross-reactivity among anti-GXM mAbs and whose binding correlates with the protective potential of mAbs in experimental infection. P601E is a mimetic, since it competes for GXM binding to 2H1, but not a mimotope, since it does not elicit an anti-GXM response. Sequence analysis of 14 anti-P601E mAbs indicates that anti-P601E mAbs elicited in BALB/c mice have an order of homology with 2H1 of V kappa > J kappa >> V(H) > J(H) > D. Further screening of a peptide library with anti-P601E mAbs isolated peptides having a motif almost identical to the peptide motif selected by 2H1. When these results are compared to the crystal structure of a related peptide in complex with 2H1, there is a clear correlation between the ability to elicit V region components of 2H1 Ab and peptide association with the V region, suggesting that the completeness of the fit in the binding site is an important driving force for mimicry. As a consequence, improving affinity of a mimetic for the Ab binding site seems to be the most logical way to insure that all of the appropriate V region segments are elicited and that useful mimotopes are created.

Somatic hypermutation of antibody genes: a hot spot warms up.

In the course of an immune response, antibodies undergo affinity maturation in order to increase their efficiency in neutralizing foreign invaders. Affinity maturation occurs by the introduction of multiple point mutations in the variable region gene that encodes the antigen binding site. This somatic hypermutation is restricted to immunoglobulin genes and occurs at very high rates. The precise molecular basis of this process remains obscure. However, recent studies using a variety of in vivo and in vitro systems have revealed important regulatory regions, base motifs that are preferred targets of mutation and evidence that transcription may play an active role in hypermutation.

Characterization of a murine monoclonal antibody to Cryptococcus neoformans polysaccharide that is a candidate for human therapeutic studies.

The murine monoclonal antibody (MAb) 18B7 [immunoglobulin G1(kappa)] is in preclinical development for treatment of Cryptococcus neoformans infections. In anticipation of its use in humans, we defined the serological and biological properties of MAb 18B7 in detail. Structural comparison to the related protective MAb 2H1 revealed conservation of the antigen binding site despite several amino acid differences. MAb 18B7 was shown by immunofluorescence and agglutination studies to bind to all four serotypes of C. neoformans, opsonize C. neoformans serotypes A and D, enhance human and mouse effector cell antifungal activity, and activate the complement pathway leading to deposition of complement component 3 (C3) on the cryptococcal capsule. Administration of MAb 18B7 to mice led to rapid clearance of serum cryptococcal antigen and deposition in the liver and spleen. Immunohistochemical studies revealed that MAb 18B7 bound to capsular glucuronoxylomannan in infected mouse tissues. No reactivity of MAb 18B7 with normal human, rat, or mouse tissues was detected. The results show that both the variable and constant regions of MAb 18B7 are biologically functional and support the use of this MAb in human therapeutic trials.

Immunoglobulin hypermutation in cultured cells.

Studies of endogenous and engineered Ig genes in mice have begun to reveal some of the cis-acting regions that are involved in the somatic hypermutation of variable regions in vivo. These studies suggest that the initiation of transcription plays a role in this process. However, it will be difficult to identify and manipulate the individual genetic elements and the trans-acting proteins that regulate and target the mutational events using solely in vivo assays. These studies would be greatly facilitated if constructs containing the genetic elements that are essential for V-region mutation could be transfected into cultured cells and undergo high rates of V-region mutation in vitro, and if permissive and non-permissive cell lines could be identified. Such in vitro systems would also allow a detailed molecular and biochemical analysis of this process. Here, we discuss some of the in vitro systems that have been developed and use data from our own studies in cultured cells to illustrate the potential benefits of studying V-region hypermutation in model in vitro systems.

Antibody-mediated modulation of Cryptococcus neoformans infection is dependent on distinct Fc receptor functions and IgG subclasses.

Coupling of an antibody response to effector cells through the Fc region of antibodies is a fundamental objective of effective vaccination. We have explored the role of the Fc receptor system in a murine model of Cryptococcus neoformans protection by infecting mice deleted for the common gamma chain of FcRs. Passive administration of an IgG1 mAb protects FcRgamma+/- mice infected with C. neoformans, but fails to protect FcRgamma-/- mice, indicating that the gamma chain acting through FcgammaRI and/or III is essential for IgG1-mediated protection. In contrast, passive administration of an IgG3 mAb with identical specificity resulted in enhanced pathogenicity in gamma chain-deficient and wild-type mice. In vitro studies with isolated macrophages demonstrate that IgG1-, IgG2a-, and IgG2b-opsonized C. neoformans are not phagocytosed or arrested in their growth in the absence of the FcRgamma chain. In contrast, opsonization of C. neoformans by IgG3 does not require the presence of the gamma chain or of FcRII, and the internalization of IgG3-treated organisms does not arrest fungal growth.

Isotype switching increases efficacy of antibody protection against Cryptococcus neoformans infection in mice.

The isotype and epitope specificities of antibodies both contribute to the efficacy of antibodies that mediate immunity to Cryptococcus neoformans, but the relationship between these properties is only partially understood. In this study, we analyzed the efficacy of protection of two sets of immunoglobulin G (IgG) isotype switch variants from two IgG3 monoclonal antibodies (MAbs) which are either not protective or disease enhancing, depending on the mouse model used. The two IgG3 MAbs 3E5 and 4H3 have different epitope specificities. Protection experiments were done with A/JCr mice infected intravenously with C. neoformans and administered with 3E5 IgG3 and its IgG1, IgG2a, and IgG2b switch variants. These experiments revealed that IgG1, IgG2b, and IgG2a were each more effective than IgG3. For 4H3 IgG3 and its IgG1 and IgG2b switch variants, the relative efficacy was IgG2b > IgG1 >> IgG3. The combination of 3E5 IgG3 and 4H3 IgG3 was more deleterious than either IgG3 alone. All IgG isotypes were opsonic for mouse bronchoalveolar cells, with the relative efficacy being IgG2b > IgG2a > IgG1 > IgG3. These results (i) confirm that a nonprotective IgG3 MAb can be converted to a protective MAb by isotype switching, (ii) indicate that the efficacy of protection of an IgG1 MAb can be increased by isotype switching to another subclass, (iii) show that protective and nonprotective IgG MAbs are opsonic, and (iv) provide additional evidence for the concept that the efficacy of the antibody response to C. neoformans is dependent on the type of MAb elicited.

Sequence dependent hypermutation of the immunoglobulin heavy chain in cultured B cells.

The variable (V) regions of immunoglobulin heavy and light chains undergo high rates of somatic mutation during the immune response. Although point mutations accumulate throughout the V regions and their immediate flanking sequences, analysis of large numbers of mutations that have arisen in vivo reveal that the triplet AGC appears to be most susceptible to mutation. We have stably transfected B cell lines with gamma2a heavy chain constructs containing TAG nonsense codons in their V regions that are part of either a putative (T)AGC hot spot or a (T)AGA non-hot spot motif. Using an ELISA spot assay to detect revertants and fluctuation analysis to determine rates of mutation, the rate of reversion of the TAG nonsense codon has been determined for different motifs in different parts of the V region. In the NSO plasma cell line, the (T)AGC hot spot motif mutates at rates of approximately 6 x 10(-4)/bp per generation and approximately 3 x 10(-5)/bp per generation at residues 38 and 94 in the V region. At each of these locations, the (T)AGC hot spot motif is 20-30 times more likely to undergo mutation than the (T)AGA non-hot spot motif. Moreover, the AGA non-hot spot motif mutates at as high a rate as the hot spot motif when it is located adjacent to hot spot motifs, suggesting that more extended sequences influence susceptibility to mutation.