Edward J Steele

PCR AMPLIFICATION OF MURINE IMMUNOGLOBULIN GERMLINE V GENES : STRATEGIES FOR MINIMIZATION OF RECOMBINATION ARTEFACTS

Murine immunoglobulin germline V genes exist as multiple sequences arranged in tandem in germline DNA. Because members of V gene families are very similar, they can be amplified simultaneously using the polymerase chain reaction (PCR) with a single set of primers designed over regions of sequence similarity. In the present paper, the variables relevant to production of artefacts by recombination between different germline sequences during amplification are investigated. Pfu or Taq DNA polymerases were used to amplify from various DNA template mixtures with varying numbers of amplification cycles. Pfu generated a higher percentage of recombination artefacts than Taq. The number of artefacts and their complexity increased with the number of amplification cycles, becoming a high proportion of the total number of PCR products once the ‘plateau phase’ of the reaction was reached. Recombination events were located throughout the ∼ 1‐kb product, with no preferred sites of cross‐over. By using the minimally detectable PCR bands (produced by the minimum number of amplification cycles), recombination artefacts can be virtually eliminated from PCR amplifications involving mixtures of very similar sequences. This information is relevant to all studies involving PCR amplification of members of highly homologous multigene families of cellular or viral origin.

The adjuvanticity of gamma inulin.

Gamma‐inulin (g‐IN) is a polymorph identified as the active component of inulin preparations that specifically activates the alternative pathway of complement (APC). The APC is central to many leucocyte functions, including B cell activation. We show here that g‐IN, when formulated as a pure, endotoxin‐free, fine suspension insoluble at 37°C and given at 50–100 μg per mouse, is a potent adjuvant for both humoral and cell‐mediated responses to a variety of antigens. g‐IN increased secondary IgG responses five‐ to 28‐fold (P <0·001), using as antigen phosphorylcholine coupled to keyhole limpet haemocyanin; subclasses IgG 2a, 2b, and 3 were boosted several hundred‐fold, IgG 1 10‐fold. IgM and IgA were increased four‐to six‐fold. Delayed hypersensitivity, by footpad swelling after secondary challenge with sheep red blood cells (SRBC), was increased more than two‐fold (P <0·001) if g‐IN was included with the primary SRBC, equivalent to increasing primary doses 10‐fold, g‐IN was equally active if given 5 days before the primary SRBC. Thus it is an immune stimulant rather than a depot or vehicle for antigen. Mice primed subcutaneously with 30–300 HA units of H2N2 influenza virus (strain A/JAP) and challenged intranasally with a lethal dose of HIN1 virus (strain A/WSN) all died, but if g‐IN was given with the primary antigen 50% of the mice survived(P < 0·001), a deduced but not proven boost to cytotoxic T cell‐mediated immunity. Unpublished work has shown that g‐IN has no adverse effects at adjuvant‐active doses. g‐IN is thus a promising new vaccine adjuvant. It also has a potential for antitumour therapy, and is a specific reagent for expioring the role of complement in vivo.

The adjuvanticity of Algammulin, a new vaccine adjuvant

Algammulin is a suspension of 1-2 microns ovoids of the immune stimulant gamma inulin (g-IN) in which alum is embedded as a carrier for protein or other anionic antigens. Tests for specific IgG and seroconversion responses in mice immunized with keyhole limpet haemocyanin (KLH) show that the presence of g-IN on the alum has increased its adjuvanticity 6- to 17-fold (p less than 0.001), and thus has a synergistic effect. A mixture of alum and g-IN was no more active than alum alone. Low KLH doses at borderline seroconversion levels (0.1-1 microgram/mouse) allow comparison with a vaccine situation. The improved Algammulin responses extended to 58 days after primary doses and to memory recall after boost at 65 days. At 1 mg Algammulin i.p. in primary and secondary doses the anti-KLH IgG responses were equal to those from Freunds complete adjuvant. The g-IN on the alum increased all responses tested (IgG 1, 2a, 2b, 3 and total IgG, IgM, IgA and IgE), but changed the emphasis from that of alum (favouring mostly IgG 2a, IgG 2b and IgA).

Algammulin, a new vaccine adjuvant comprising gamma inulin particles containing alum: preparation and in vitro properties

Crystallization of inulin with alum forms a fine (1-2 micron) suspension of electron-dense ovoids; the alum is embedded in inulin particles, which are then converted to the immune stimulant polymorphic form, gamma inulin. This very stable hybrid preparation is termed Algammulin. Preferred conditions for its preparation are described. The alum still adsorbs protein. Gamma inulin is equally able to activate the alternative pathway of complement in vitro whether free or combined as Algammulin. Gamma inulin, either free or combined as Algammulin, dissolved on heating over a narrow temperature range that can be used to characterize the polymorphic form of the inulin.

Human DNA polymerase-η, an A-T mutator in somatic hypermutation of rearranged immunoglobulin genes, is a reverse transcriptase

We have proposed previously that error‐prone reverse transcription using pre‐mRNA of rearranged immunoglobulin variable (IgV) regions as templates is involved in the antibody diversifying mechanism of somatic hypermutation (SHM). As patients deficient in DNA polymerase‐η exhibit an abnormal spectrum of SHM, we postulated that this recently discovered Y‐family polymerase is a reverse transcriptase (RT). This possibility was tested using a product‐enhanced RT (PERT) assay that uses a real time PCR step with a fluorescent probe to detect cDNA products of at least 27−37 nucleotides. Human pol‐η and two other Y‐family enzymes that are dispensable for SHM, human pols‐ι and ‐κ, copied a heteropolymeric DNA‐primed RNA template in vitro under conditions with substantial excesses of template. Repeated experiments gave highly reproducible results. The RT activity detected using one aliquot of human pol‐η was confirmed using a second sample from an independent source. Human DNA pols‐β and ‐µ, and T4 DNA polymerase repeatedly demonstrated no RT activity. Pol‐η was the most efficient RT of the Y‐family enzymes assayed but was much less efficient than an HIV‐RT standard in vitro. It is thus feasible that pol‐η acts as both a RNA‐ and a DNA‐dependent DNA polymerase in SHM in vivo, and that Y‐family RT activity participates in other mechanisms of physiological importance.

Mechanism of somatic hypermutation: Critical analysis of strand biased mutation signatures at A:T and G:C base pairs

The DNA sequence data of the somatic hypermutation (SHM) field published since 1984 has been critically reviewed. The analysis has revealed three strand biased mutation signatures. The first concerns the mutations generated at G:C base pairs in mice genetically deficient in uracil-DNA glycosylase and MSH2-MSH6-mediated mismatch repair. Such mice display the AID deaminase footprint and here C mutations exceed G mutations at least 1.5-fold. This supports earlier and more recent studies claiming that dC-to-dU deaminations occur preferentially in the single stranded DNA regions of the displaced nontranscribed strand (NTS) during transcription. The second concerns the signature generated in immunised mice where G mutations exceed C mutations by at least 1.7-fold. This is a newly identified strand bias which has previously gone undetected. It is consistent with the polynucleotide polymerisation signature of RNA polymerase II copying the template DNA strand carrying AID-mediated lesions generated at C bases, viz. uracils and abasic sites. A reverse transcription step would then need to intervene to fix the mutation pattern in DNA. The third concerns the long recognised strand biased signature generated in normal aged or actively immunised mice whereby A mutations exceed T mutations by two- to three-fold. It is argued that this pattern is best understood as a combination of adenosine-to-inosine (A-to-I) RNA editing followed by a reverse transcription step fixing the A-to-G, as well as A-to-T and A-to-C, as strand biased mutation signatures in DNA. The reasons why the AID-linked RNA polymerase II mutation signature had previously gone undetected are discussed with regard to limitations of standard PCR-based SHM assay techniques. It is concluded that the most economical SHM mechanism involves both DNA and RNA deaminations coupled to a reverse transcription process, most likely involving DNA polymerase eta acting in its reverse transcriptase mode. Experimental approaches to differentiate this RNA-based model from the standard DNA deamination model are discussed.

The signature of somatic hypermutation appears to be written into the germline IgV segment repertoire

Summary: We present here a unifying hypothesis for the molecular mechanism of somatic hypermutation and somatic gene conversion in IgV genes involving reverse transcription using RNA templates from the V‐gene loci to produce cDNA which undergoes homologous recombination with chromosomal V(D)J DNA. Experimental evidence produced over the last 20 years is essentially consistent with this hypothesis. We also review evidence suggesting that somatically generated lgV sequences from B lymphocytes have been fed back to germline DNA over evolutionary time.

MECHANISM OF ANTIGEN-DRIVEN SOMATIC HYPERMUTATION OF REARRANGED IMMUNOGLOBULIN V(D)J GENES IN THE MOUSE

Available data relevant to the mechanism of somatic hypermutation have been critically evaluated in the context of alternative models: (i) error‐generating reverse transcription (RT) followed by homologous recombination: and (ii) error‐prone DNA replication/repair. A set of basic principles concerning somatic hypermutation has also been formulated and a revised and expanded ‘RT‐Mutatorsome” concept (analogous to telomerase) is presented which is consistent with these principles and all data on the distribution of somatic mutations in normal and Ig transgenic mice carrying particular V(D)J and flanking region constructs. It is predicted that in the mouse VH and Vk loci, the J–C intronic Enhancer‐Nuclear Matrix Attachment Region (Ei/MAR) contains a unique sequence motif or secondary structure which ensures that only V(D)J sequences mutate whilst other regions of the genome are not mutated.

Genesis of the strand-biased signature in somatic hypermutation of rearranged immunoglobulin variable genes.

The history and current development of the reverse transcriptase model of somatic hypermutation (RT‐model) is reviewed with particular reference to the genesis of strand‐biased mutation signatures in rearranged immunoglobulin variable genes (V(D)J). The recent disagreement in the field as to whether strand bias really exists or not has been critically analysed and the confusion traced to the putative presence, in some mutated V(D)J sequence collections, of polymerase chain reaction (PCR)‐recombinant artefacts. Recent analysis of somatic hypermutation in xeroderma pigmentosum variant patients, by the group of PJ Gearhart and others, has established that the Y‐family translesion DNA repair enzyme, DNA polymerase η (eta), is responsible for the striking A‐T targeted strand‐bias mutation signature seen in all mouse and human collections of somatically mutated V(D)J sequences. This evidence, together with our own recent demonstration that human DNA polymerase η is a reverse transcriptase, leads to the conclusion that the strand‐biased A‐T mutation signature is caused either by: (i) error‐prone DNA‐dependent DNA repair synthesis by pol‐η of single‐strand nicks preferentially in the non‐transcribed strand; and/or (ii) by error‐prone cDNA synthesis of the transcribed strand by pol‐η using the pre‐mRNA as the copying template, primed by the nicked transcribed DNA strand, followed by replacement of the original transcribed strand by cDNA. Analysis of the total mutation pattern also suggests that the major transitions observed in SHM (A→G, C→T and G→A) can be explained by known RNA editing mechanisms active on pre‐mRNA which are then written into cDNA during synthesis of the transcribed strand by error‐prone cellular reverse transcriptases such as pol‐η.

Affinity maturation of lymphocyte receptors and positive selection of T cells in the thymus

In this review we have re-evaluated the dominant paradigm that TcR V genes do not somatically mutate. We highlight the many structural and functional similarities between Ig and TcR antigen-specific receptors on B and T cells. We have reviewed the factors influencing the somatic and germline evolution of IgV regions in B cells, have evaluated in detail various models which could be invoked to explain the pattern of variation in both transcribed and non-transcribed segments of germline IgV-gene DNA sequences, and applied this perspective to the TcR V beta and V alpha genes. Whilst specific TcRs recognize a complex of a short antigenic peptide bound to MHC Class I or II glycoprotein, and Ig receptors can recognize both oligopeptides and conformational determinants on undegraded polypeptides, they both employ heterodimer variable regions (Fabs) utilizing all three CDRs in epitope binding. We conclude that a plausible case can be made for the possibility that rearranged TcR V genes may undergo some type of somatic hypermutation process during T-cell development in the thymus (concurrent with or after the positive selection phase) thus allowing a repertoire of TvR alpha beta heterodimers to be both positively and negatively selected by the same set of ligands (self MHC + self peptide) in the thymus.