Peter McCullagh

A New Model of Sheep Ig Diversification: Shifting the Emphasis Toward Combinatorial Mechanisms and Away from Hypermutation

The current model of Ig repertoire development in sheep focuses on the rearrangement of a small number (∼20) of Vλ gene segments. It is believed that this limited combinatorial repertoire is then further diversified through postrearrangement somatic hypermutation. This process has been reported to introduce as many as 110 mutations/1000 nucleotides. In contrast, our data have that indicated somatic hypermutation may diversify the preimmune repertoire to a much lesser extent. We have identified 64 new Vλ gene segments within the rearranged Ig repertoire. As a result, many of the unique nucleotide patterns thought to be the product of somatic hypermutation are actually hard-coded within the germline. We suggest that combinatorial rearrangement makes a much larger contribution, and somatic hypermutation makes a much smaller contribution to the generation of diversity within the sheep Ig repertoire than is currently acknowledged.

The significance of immune suppression in normal self tolerance.

The most immediate requirement of research relating to dominant immunological tolerance is to clarify its significance relative to other mechanisms for the functioning of the immune system. A similar imperative applies in relation to all of the other mechanisms that have heen claimed to mediate tolerance. Several decades of research on immunological tolerance have abundantly indicated what can happen: it is necessary now to identify what most commonly does happen and the circumstances predisposing to this. In attempting a synthesis that could explain the significance of the apparently diverse mechanisms that have heen demonstrated to account for self tolerance, I propose to a.ssume., as a first principle, that all represent aspects of one integrated system. It appears inherently implausihle to me that fragments of a numher of altemative ways of ensuring self tolerance would remain operative and independently functioning. My second assumption is that examination of the evolutionary development of immunological tolerance is required to attain an understanding of the significance of the processes that are responsible for self tolerance in mammals. The confidence with which immunological tolerance of self could he regarded as understood has steadily diminished as experimental data relating to it has proliferated. One suspects that the first major confusion about the nature of immunological tolerance arose hecause of the temporal proximity between the prediction of induc-

Transient transmission of porcine endogenous retrovirus to fetal lambs after pig islet tissue xenotransplantation

Evidence for the in vivo transmission of porcine endogenous retrovirus (PERV) from porcine xenografts to various recipient animals has been inconsistent. To characterize the contribution of the host immune system to the potential for PERV transmission from pig islet tissue xenografts to host tissues, we examined two immunoincompetent animal models, thymectomizsed fetal lambs and NODscid mice. Pig proislets were grafted into fetal lambs or adult NODscid mice. Conventional, nested and real‐time PCR/RT‐PCR tests were used to search for PERV and pig cell‐specific sequences (porcine mitochondrial cytochrome oxidase II (COII) or mitochondrial ribosomal 12S) in pig proislets, host liver and spleen at 5–84 days (lambs) or 96 days (mice) after transplantation. Xenografts were harvested at the same time points. The copy number of PERV sequences and host cell‐specific nuclear (palmitoylcarnitine transferase) sequences was assessed by real‐time PCR to estimate the proportion of PERV‐infected host cells. Pig proislets were shown to be PERV+ve by PCR and immunohistochemistry (PERV B env protein p15E). PERV transmission (PERV A, B or C DNA in the absence of porcine COII or 12S sequences) was detected by nested PCR and real‐time PCR in 4/12 fetal lamb liver samples 5–23 days after transplantation; the maximum copy number of PERV B env sequences was found at day 5 (700 copies/1 × 106 lamb cells). A total of 4/12 fetal lambs demonstrated both PERV and 12S porcine sequences in liver samples (days 5–84) by real‐time PCR, suggesting that pig cells had migrated to those tissues and established microchimerism; nested PCR showed evidence for microchimerism (porcine COII sequences alone) in 2/12 lambs (day 5). The incidence of PERV transmission and frequency of microchimerism was similar in host spleen analysed by real‐time PCR. Histological examination showed complete xenograft rejection by 23 days after transplantation to fetal lambs. In contrast, pig proislet xenografts survived long term (⩾day 96) in NODscid mice but no PERV transmission was found. Both nested and real‐time PCR assays revealed that 2/3 mice had become microchimeric. Long‐term expression of PERV A, B and C as well as porcine 12S or COII RNAs was found at the graft site (day 96) only, indicating that PERV transcription and possibly replication, continued in the donor pig islet tissue after transplantation. Overall, detection of PERV transmission and microchimerism was limited by the sensitivity of the PCR assay and the primers chosen. The absence of stable PERV transmission and microchimerism in fetal lambs and the rejection of pig proislet xenografts correlated in time with the establishment of host immunocompetence. We therefore suggest that the frequent failure to identify PERV transmission late after transplantation could be due to the immunological destruction of PERV‐infected host cells. Recipient NODscid mice demonstrated long‐term microchimerism and intragraft PERV expression, which was consistent with their stable immunoincompetence.

Effect of early fetal splenectomy on prenatal B-cell development in sheep

The contribution of early splenic B‐cell populations to the colonization of the ileal Peyers patch was investigated following the surgical removal of the spleen in a series of 56‐day‐old fetal sheep. The fetuses were killed at 140 days of gestation and the ileal Peyers patch, the distal jejunal lymph node which drains the Peyers patch, and a peripheral lymph node, the superficial cervical lymph node, were examined. Enzyme and immunohistochemical evaluation concluded that the distribution of B cells, T cells and stromal cells in the ileal Peyers patch was similar in splenectomized and normal fetal sheep. Thus, the presence of the fetal spleen was not essential for the colonization of the ileal Peyers patch and other early sites of B‐cell accumulation would appear capable of generating the necessary precursor populations. Investigation of B‐cell populations in lymph nodes used a combination of terminal deoxynucleotidyl‐transferase‐mediated deoxyuridine‐triphosphate nick‐end‐labelling (TUNEL) histochemistry and immunofluorescence to determine the average number of apoptotic B cells in the primary follicles of the outer cortex of splenectomized and normal lambs. A significantly increased number of apoptotic B cells was present in the distal jejunal lymph node but not in the superficial cervical lymph node of splenectomized lambs. This finding suggests that splenectomy affected prenatal B‐cell development in fetal sheep and raises questions as to the regulation of B‐cell lymphopoiesis in a species using a post‐rearrangement organ of diversification.

Immunological responsiveness of maternal and foetal lymphocytes during normal pregnancy in the ewe

Peripheral blood lymphocytes collected from ewes before and during pregnancy manifested constant reactivity to concanavalin A and to paternal and third party peripheral blood lymphocytes. However, in some instances, the reactivity of these maternal cells against lipopolysaccharide and lymphatic lymphocytes from paternal, foetal and third-party donors increased markedly during pregnancy. Apart from an indication that plasma from some pregnant ewes acquired the capacity to depress lymphocyte reactivity non-specifically, no evidence was obtained to suggest that maternal lymphocyte reactivity observed in vitro did not accurately reflect the capacity of these cells in the donor ewe. In particular, there was no indication that populations of maternal peripheral blood lymphocytes returning from the gravid uterus had undergone any modification of reactivity against foetal determinants.

Suppression of anti-thyrocyte autoreactivity by the lymphocytes of normal fetal lambs

We have devised an experimental strategy to determine whether the developing immune system of normal fetal animals can spontaneously acquire the capacity to inhibit autoimmune responses by its cells as it matures. Whilst the existence of cells with the capacity to exert negative regulation and to curtail autoimmune responses has been demonstrated previously in response to the experimental induction of these responses, the relevance of such regulatory processes to the prevention of overt autoimmunity in normal animals has not been established. We have produced pairs of identical twin fetal lambs by splitting blastocysts and have subsequently deprived one of each pair of exposure to thyroid-specific antigens by surgical thyroidectomy before development of immunological self recognition. Thyroidectomized fetuses developed T lymphocytes autoreactive against self thyrocytes. However, their normal, identical co-twins were found to acquire a class of T lymphocytes with the capacity to block anti-thyrocyte autoreactive cells from the thyroidectomized fetal co-twin. Blocking of anti-thyroid autoreactivity required preliminary contact between these normal T lymphocytes and the target thyrocytes. Substitution of an allograft of fetal thyroid tissue for a fetal lambs own thyroid gland failed to prevent the development of autoreactivity against autologous thyrocytes by the recipients lymphocytes. However, the reactivity of those lymphocytes against thyrocytes from the specific allogeneic thyroid donor was markedly curtailed.

Modelling of peripheral lymphocyte migration: system identification approach.

This is the first application of the prediction error method (PEM) of system identification to modelling lymphocyte migration through peripheral lymphoid tissue. The PEM was applied to the emergence of labelled lymphocytes from the efferent lymphatic of a lymph node following their intravenous administration. Advantages of PEM included the capacity to calculate the response to a unit impulse stimulus, unavailable to direct observation, and to allow for the return to the node of labelled cells that had already recirculated once. Calculation of the system delay (time between introduction of cells into the blood and their first appearance in lymph) indicated 4.67 ± 1.05 h for the total lymphocyte population. The peak in efferent lymph occurred at 11.91 ± 4.68 h, much earlier than previous reports, which were affected by cells that had already recirculated. While 75% of labelled cells had emerged in efferent lymph by 20.77 ± 5.62 h, 86.38 ± 29.44 h was required for 100% emergence. The considerable heterogeneity in migratory behaviour is likely to reflect frequency and duration of binding of lymphocytes by dendritic cells in paracortical cord corridors. It is proposed that differences in the speed with which lymphocytes pass along corridors depend on their functional status, in particular whether they are naïve or memory cells.

The response of the foetal lamb to maternal lymphocytes

Foetal lambs were inoculated with either maternal or third-party lymphocytes. Of foetuses transfused in the first half of pregnancy (from 49 to 73 days), one quarter survived until the fifth month. Examination of the immunological reactivity of these survivors revealed that all rejected skin grafts from the lymphocyte donors and manifested normal mixed lymphocyte reactivity. In two instances, responsiveness of the transfused lambs to normal lymphocyte transfer was reduced. Foetal lambs transfused with large numbers of maternal lymphocytes in the last third of pregnancy could survive provided the donor ewe had not been sensitized against foetal or paternal determinants. Following intravenous challenge with maternal lymphocytes, cells collected over a prolonged period from the thoracic duct of the foetal recipient exhibited depression of anti-maternal reactivity in mixed lymphocyte culture.

B-cell development: one problem, multiple solutions.

Interspecies variations in the processes of B‐cell development and repertoire generation contrast with the greater consistency of T‐cell development. B‐cell development in mice and humans, with postnatal B‐cell generation of new repertoire in the bone marrow throughout life, is regarded as the ‘standard’ pattern. In contrast, accounts of B cells in birds, sheep, cattle, rabbits and pigs (the ‘other’ species) describe cessation of gene diversification in the perinatal period, with the gut‐associated lymphoid tissue (GALT) functioning as the primary lymphoid organ thereafter. It has become customary to regard the developmental pathways of T and B cells within any individual species as being as dissimilar as the functions of the two mature cell types. Reinterpretation of B‐cell development patterns in different species is overdue in response to two types of reports. The first of these describe T–B ‘crossover’, specifically the intrathymic production of B cells and the extrathymic production of T cells. The second attests to the extent of sharing of B‐cell developmental features across the two groups of species. We propose that, as is a feature of other haematopoietic cells, a menu of alternative B‐ and T‐cell pathways has been retained and shared across species. A single pathway usually predominates in any species, masking alternatives. The observed predominance of any pathway is determined by factors such as placental permeability, extent of maturation of the immune system by birth and the feasibility of direct experimental intervention in development.

Development of B cells in the gut-associated lymphoid tissue of mid-gestational fetal lambs

Intestinal mucosal immune system development was investigated in fetal lambs from 61 to 110 days (term, 150 days). Fetal small intestine was examined at 400 mm intervals for the presence of IgM(+) cells. The first phase of B cell development was characterised by increase in B cell density throughout the jejunum from 65 days. Increase in density was greatest in the proximal jejunum and declined progressively approaching the ileum. The second phase entailed a decrease in jejunal B cell concentration, evident from 90 days. The average number of cells per field diminished, by 110 days, to a 10th that at 90 days. Failure of B cell increase to match a five-fold intestinal lengthening may have contributed to this. Overlapping the two phases of jejunal B cell development was a third phase of major expansion of B cell density in the terminal ileum.