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Ageing is a process characterised by progressive loss of function in multiple different organ systems, such as the nervous, endocrine and immune systems. Current data showing that ageing processes may be associated with alterations in the immune system suggest that some of the genetic determinants of senescence might be polymorphic genes that regulate immune responses. The 'Immunogenetics of Aging' programme was a component introduced in the 14th International HLA and Immunogenetics Workshop ( IHIWS) and developed further within the 15th and 16th. The aim of this component was to determine the contribution of immune genes to successful ageing and an increased capacity to reach the extreme limits of lifespan. Within the 16th IHIWS, new populations were included, and the number of samples analysed was increased. Analysis was focused on innate immunity genes ( KIR and MBL2) and their correlation with CMV serostatus. Collaborative studies suggested that both activating and inhibitory KIR and functionally relevant MBL2 haplotypes are important factors for control of CMV infection in the elderly and therefore for chronic low-grade inflammation. Results showed that these genes might be predictive biomarkers in ageing and longevity. Prevalence of MBL2 haplotypes determining absence of the protein ( LYPB, LYQC and HYPD) was observed in elderly people with a higher CMV antibody titre. The high CMV titre was also associated with a decreased frequency of the activatory KIR2 DS5 and A1 B10 haplotypes in elderly. Due to the role of KIR and low or deficient MBL haplotypes in viral infections, these genetic markers could be considered as indicators of a need for CMV prophylaxis at younger age and therefore increased probability of longer lifespan. [ABSTRACT FROM AUTHOR]

An age-related decline in immune responses in the elderly results in greater susceptibility to infection and reduced responses to vaccination. This decline in immune function affects both innate and adaptive immune systems. A meeting of experts in immunology and gerontology in Paris, France, in April 2008, considered current understanding of immunosenescence and its clinical consequences. Essential features of immunosenescence include: reduced natural killer cell cytotoxicity on a per cell basis; reduced number and function of dendritic cells in blood; decreased pools of naive T and B cells; and increases in the number of memory and effector T and B cells. In particular, an accumulation of late differentiated effector T cells, commonly associated with cytomegalovirus infection, contributes to a decline in the capacity of the adaptive immune system to respond to novel antigens. Consequently, vaccine responsiveness is compromised in the elderly, especially frail patients. Strategies to address the effects of immunosenescence include ensuring that seroprotective antibody levels against preventable infectious diseases are maintained throughout adulthood, and improving diet and exercise to address the effects of frailty. New vaccines are being developed, such as intradermal and high-dose vaccines for influenza, to improve the efficacy of immunization in the elderly. In the future, the development and use of markers of immunosenescence to identify patients who may have impaired responses to vaccination, as well as the use of end-points other than antibody titers to assess vaccine efficacy, may help to reduce morbidity and mortality due to infections in the elderly. [ABSTRACT FROM AUTHOR]

The interpretation of the results obtained from immunomonitoring of clinical trials is a difficult task due to the variety of methods and protocols available to detect vaccine-specific T-cell responses. This heterogeneity as well as the lack of standards has led to significant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer (“CIMT”) in order to compare techniques and protocols applied for the enumeration of antigen-specific T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the “CIMT monitoring panel”. A total of 13 centers from six European countries participated in the study in which pre-tested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-specific T-cells in each sample using tetramer staining and one functional assay. The results of the first testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-specific CD8+ T-cells by tetramer staining. Analysis by ELISPOT was influenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the first phase. The recommendations improved the number of antigen-specific T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identification of distinct variables that influence the sensitivity of different T-cell assays and to formally show that a defined correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such “two-step” inter-laboratory projects could define rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring. [ABSTRACT FROM AUTHOR]

An age-related accumulation of DNA damage caused by increased insult and/or decreased repair, could contribute to impaired cellular function. DNA mismatch repair (MMR), the main postreplicative correction pathway, can be monitored by assessing microsatellite instability and has been reported to decrease with age. Here, we analyzed the involvement of the MMR system in the accumulation of genetic damage in a cultured monoclonal human T lymphocyte model. We correlated microsatellite instability (MSI) and MMR gene expression, and replicative senescence of CD4+ clones derived from young, old and centenarian individuals or from CD34+ precursors. Cells were analyzed for MSI at five loci (CD4, VWA, Fes, D2S123, and BAT26), for the methylation status of MLH1 and MSH2 gene promoters, and for the expression of the MMR genes MSH2, MSH6, MSH3, MLH1, PMS2, and PMS1. MSI increased with increasing culture passages, particularly in the CD34+ progenitor-derived clones, but also in those from adult T cells. MSI and MMR gene expression were found to correlate, mostly due to a reduced expression of the components of MutL heterodimers, pointing to a role of MMR in the acquisition of DNA damage with in vitro aging. [ABSTRACT FROM AUTHOR]

The rate of acceleration of the frequency of death due to cardiovascular disease or cancer increases with age from middle age up to around 75–80 years, plateauing thereafter. Mortality due to infectious disease, however, does not plateau, but continues to accelerate indefinitely. The elderly are particularly susceptible to novel infectious agents such as SARS, as well as to previously encountered pathogens. Why is this? The elderly commonly possess oligoclonal expansions of T cells, especially of CD8 cells, which, surprisingly, are associated with cytomegalovirus (CMV) seropositivity. This in turn is associated with many of the same phenotypic and functional alterations to T cell immunity that have been suggested as biomarkers of immune system aging. We suggest that, in fact, CMV, not age per se, is the prime driving force behind many or most of the oligoclonal expansions and altered phenotypes and functions of CD8 cells in the elderly. Thus, the manner in which CMV and the host immune system interact (over which period? on which genetic background? with which co-infections?) is critical in determining the “age” of adaptive immunity and hence human longevity. In this respect, immunosenescence is infectious. [ABSTRACT FROM AUTHOR]

The effects of the immunosuppressive sulphated polygalactan λ carrageenan on in vitro models of allograft immunity were compared with the effects of removing macrophages (surface adherent and/or phagocytic cells) by established methods. Carrageenan depressed the primary mixed lymphocyte reactions, but not to the same extent as the removal of macrophages. 2-Mercaptoethanol restored the response. Secondary mixed lymphocyte reactions and responses to phytohaemaglutinin were depressed by carrageenan but not by the removal of macrophages, and in these systems 2-mercaptoethanol failed to restore the responses of carrageenan-treated cultures. In contrast, cell-mediated cytolysis by presensitized lymphocytes was not affected by carrageenan or by colloidal silica. Carrageenan depressed cell-mediated cytolysis only if it was present during the sensitization of the effector cells. We conclude that carrageenan can have two dose-related effects in vitro: one on the macrophage and one on the responding lymphocyte. [ABSTRACT FROM AUTHOR]

Regulation of the induction of suppressive activity in peripheral blood mononuclear cells (PBMC) by human major histocompatibility complex (MHC) class II+ CD4+ CD45R-suppressor-inducer T-cell clones has been investigated. Previously, it was shown that in this system, cyclosporin A-sensitive precursors gave rise to allo-indifferent MHC-unrestricted CD4+ suppressive cells. Their induction could be blocked by monoclonal antibodies (mAb) to multilocus MHC class II gene products (TÜ39) but not by mAb preferentially reacting with HLA-DR, -DQ or -DP molecules. This product, functionally defined, was termed 'DY'. It is shown here that induction of suppression by DY follows established activation pathways: (i) cell adhesion was required because CD 11a (LFA-1 ) mAb blocked suppressor-induction; (ii) CD4 mAb also blocked, consistent with the involvement of class II products in suppressor-induction; (iii) cell proliferation was required because mAb to transferrin receptors, or irradiation, inhibited induction; and (iv) such proliferation appeared to be interleukin (IL)-2-dependent because it was blocked by mAb to IL-2 receptor, and enhanced by exogenous IL-2 but not IL-4. It was also enhanced by exogenous IL-I and IL-6, but not by IL-3, tumour necrosis factor-alpha (TNFα) or interferon-gamma (IFN-γ). It therefore seems that the requirements for activation of suppression by CD4+ DY+ T-cell clones in this in vitro model bear many similarities to those for CD4+ helper T cells, namely, mediation by MHC class II with CD4 involvement, dependency on LFA-1-influenced cell interactions, and reliance on clonal expansion caused by IL-2 and possibly amplified by IL-1 and/or IL-6. [ABSTRACT FROM AUTHOR]

T-lymphocyte clones derived from populations sensitized to alloantigens in vitro were tested for their regulatory effects on pokeweed mitogen-stimulated immunoglobulin (Ig) secretion. Clones with natural killer (NK)-like cytotoxicity and/or suppressive activity for lymphoproliferative (LP) responses potently inhibited Ig secretion. Moreover, certain alloproliferative T4 + interleukin-2 (IL-2)-secreting 'helper' clones shared this strong suppressive activity on Ig secretion. The remaining clones enhanced, rather than suppressed, Ig production. Inhibition by all types of suppressive clones appeared not to be restricted by MHC products, since allogeneic HLA-mismatched donors were suppressed as efficiently as the autologous donor. Suppression was radio resistant, and was apparently not caused by absorption of IL-2, or cytotoxicity of the clones. Suppression was still detectable at plateau levels when cloned cells were added as late as 96 hr after the initiation of the cultures, suggesting an inhibitory mechanism divorced from early B-cell activation events. Thus, T-lymphocyte clones with distinct different functional activities share similar profound suppressive effects on Ig secretion in vitro. [ABSTRACT FROM AUTHOR]

Conditions for the production of highly effective human T-cell growth factor (TCGF) and its screening in a thymidine incorporation assay are described. Culture medium supplemented with optimum concentrations of TCGF was used in bulk culture systems for the expansion of alloactivated and non-alloactivated T-cell populations. Surface marker studies of cells grown in TCGF-dependent culture showed that the majority were T cells expressing DR antigens. These cells retained their proliferative responsiveness in mixed leucocyte cultures but were unable to stimulate proliferation of normal allogeneic lymphocytes. Cultured cells which derived from mixed leucocyte cultures retained immunological specificity compared with cells not exposed to TCGF both in terms of proliferative responses caused by allogeneic cells in the absence of TCGF, and in terms of cytotoxic activity against 51Cr-labelled target cells. In addition they were capable of suppressing tritiated thymidine incorporation in mixed leucocyte cultures. These experiments show that continuous culture with TCGF can expand a variety of T-cell subpopulations with retention of distinct immunological function. [ABSTRACT FROM AUTHOR]

The human histocompatibility leucocyte antigen (HLA) class II-associated invariant chain comprises at least four polypeptides. One of these is expressed as a membrane-bound subunit and has been designated as CD74. We investigated the expression of CD74 on B- and T-cell types utilizing monoclonal antibodies (mAb) specific for thc c-terminus of CD74. All B cells tested expressed CD74 regardless of their HLA phenotypes. High and iow CD74 expressors were provisionally assigned based on quantitative variations found in some haplotypes. A mutant cell with selective deletions of class II loci DR and DQ but with retention of DP and additional class II heterodimers synthesize normal amounts of CD74. In contrast, however, a mutant with a total class II deletion had diminished expression of CD74, suggesting that at least one of the class II isotypes is essential for full constitutive expression of CD74. Major histocompatibility complex (MHC) class II negative T-cell lines and CD3+ T cell isolated from peripheral blood did not express CD74. However, allostimulated T cells which express de novo induced class II determinants also did not express CD74. Taken together, these results show that CD74 is expressed on B cells and unexpectedly is not synthesized on de novo-induced class II positive T-cell clones. [ABSTRACT FROM AUTHOR]

Human histocompatibility leucocyte antigen (HLA)-specific monoclonal antibody probes were used to determine the affinity constant, and cell-surface density of HLA class I and class II determinants. The measurements were estimated for single-cell units of B-lymphoblastoid cell line (B-LCL) and cloned activated T cells in different functional states. Each HLA subset showed unimodal affinity constant values for the interaction with the corresponding HLA-specific antibodies. Such values ranged between 2·2 × 107 M-1 (class I) and 4·0 × 107 M-1 (class II) for different histocompatibility epitopes. In both B and T cells there was a rank order of epitope expression, class I being highly expressed (5 × 106 epitopes/cell) followed by DR, DQ and DP, (1·1-3·0 × 106 epitopes/cell). Suppressive clones carrying functionally defined stimulating determinants previously designated `DY' carried similar numbers of DR, DQ and DP binding sites to DY- non-suppressive clones, but showed selective increases of class II determinants reactive with broad class II-specific antibodies. The results are discussed in the context of the functional consequences of different patterns of HLA epitope expression in immune responses. [ABSTRACT FROM AUTHOR]

Secretion of different cytokines may be an important T-cell effector mechanism for bone marrow engraftment, graft versus host disease and graft versus leukaemia effects after allogeneic bone marrow transplantation (BMT). Cytokine secretion and autocrine proliferative capacity of T-cell clones derived from leukaemia patients 3 - 6 weeks after allogeneic bone marrow transplantation were investigated. Only a minority of post-transplant T-cell clones (23/120; 19%) was capable of undergoing autocrine proliferation. By contrast, 21/65(32%) normal control clones from the marrow donors derived under the same conditions were autocrine proliferative. All clones were interleukin-2 (IL-2) responsive. A majority (12/17; 71%) of autocrine proliferating post-transplant clones secreted detectable IL-2. Compared with control clones. CD4+ T-cell clones derived early after BMT produced decreased levels of interleukin-4 (IL-4) and interleukin-6 (IL-6), whereas secretion of interleukin-3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF) showed no significant difference. The small number (n = 8) of post-transplant CD8+ clones showed decreased production of IL-3., IL-4 and IL-6 compared with control clones, but normal secretion of GM-CSF. Neither CD4+ nor CD8+ T-cell clones secreted interleukin-7 (IL-7). [ABSTRACT FROM AUTHOR]

Human lymphocytes alloactivated in vitro were cloned by limiting dilution in the presence of filler cells and interleukin 2 (IL 2)-containing supernatants of phytohaemagglutinin-stimulated lymphocytes. Clones with allospecific proliferative reactivity (PLT clones), measured by tritiated thymidine (³H-TdR) incorporation, were selected for extensive IL 2-dependent expansion. The cloned lines had finite lifespans, ranging from an estimated minimum of 28 to > 65 doublings. Function as PLT reagents, however, was retained in all cases for only an estimated 30 cell doublings. This apparent cessation of function was not caused by loss of the ability to metabolize thymidine, since lines continuing to grow for >30 doublings still incorporated ³H-TdR in the presence of IL 2. An altered requirement for stimulating antigen (number of stimulating cells), or altered response kinetics, did not contribute to loss of PLT function. Exogenous IL 2 added during restimulation to responders previously ‘rested’ overnight without IL 2 did not restore the response. Thus, under present experimental conditions, functional lifespans of cloned PLT reagents appear fixed at ∼30 cell doublings. [ABSTRACT FROM AUTHOR]

Conditions for the cloning of alloactivated human lymphocytes by limiting dilution in the presence of interleukin 2 (IL 2) and filler cells have been investigated. Cloning efficiencies were extremely high (at least 40%) when IL 2 derived from pooled peripheral blood mononuclear cells (PBM), stimulated with phytohaemogglutinin in the presence of irradiated Epstein-Barr, virus-transformed lymphoid cell lines (LCL), was used in combination with irradiated pooled PBM as filler cells. Cloning efficiencies were reduced almost twofold using autologous filler cells and were further reduced dramatically using autologous IL 2, although some clones could still be obtained. In addition, cloning efficiencies were acceptable (over 30%) when IL 2 produced spontaneously from the leukaemic cell Jurkat (M-N) was used. In contrast, sheep erythrocytes and LCL as filler cells failed to allow successful cloning, although LCL were beneficial in the expansion of cloned populations. Other factors being equal, the use of pooled PBM as filler cells and highly active preparations of IL 2 will be the method of choice for cloning functional human T lymphocytes. [ABSTRACT FROM AUTHOR]

The lymphoma cell line BJAB.B95.8.6 was gamma-irradiated to induce mutations of major histocompatibility complex (MHC) encoded genes. Cloned 'wild-type' cells were phenotyped HLA-A1, A2, B 13, 1335, Bw4, Bw6, Cw4, DR5, DRw52, DQwl, DQw3, DPw2, DPw4, GLO11, PGM32-1, and ME10 and possessed two apparently normal chromosome 6s prior to mutagenesis. Loss mutants were selected 5 days after 3 Gy gamma-irradiation employing three complement-fixing monoclonal antibodies specific for HLA-A2 (TÜ101) and Bw4 (TÜ48, TÜ109). Fifteen independently arising mutants were isolated and cloned. Typing with monospecific alloantisera and cell-mediated lympholysis revealed the presence of HLA-A1, 835, Bw6, Cw4, DR5. DRw52, DQw3, and DPw4 specificities on all mutant clones. HLA-A2, B13, and Bw4 were absent. Mutants differed in their expression of class 11 antigens. One group retained DQw1 and DPw2, another was DQw1, DPw2, and a third was DQw1, DPw2. Karyotyping of the 'wild-type' line and selected mutant clones showed that the loss of HLA specificities correlated with deletions which map the HLA-A and - B loci directly to the distal part of the 6p2l.33 region and the class II genes to the region 6p21.33 (proximal) to 6p21.31 (distal) on the short arm of chromosome 6. [ABSTRACT FROM AUTHOR]

The HLA-linked 'SB' antigens comprise a new segregant series of B-cell alloantigens mapping between HLA-DR and glyoxylase. They can be detected by secondary proliferative responses of lymphocytes primed against HLA-A, B, C, DR, MB- and MT-compatible stimulators. To assess genetic complexity of the SB-gene region, alloreactive cloned T-cell lines were derived from four reagents detecting specificities designated SB2 and SB3. In two families, products detected by seven different clones segregated with the HLA haplotypes bearing the SB2 or SB3 specificities as recognized by the uncloned reagents. There were no indications that the cloned cells differed from the oligoclonal reagents in their fine specificity. In contrast to previous results with an S134-associated specificity, in population studies of 25 S132-positive and 23 SB3-positive donors, no evidence could be found for subtypes of either specificity. Thus, even at the level of recognition by cloned T-cells, both S132 and SB3 appear to be remarkably homogeneous in the population. [ABSTRACT FROM AUTHOR]

Alloactivated human lymphocytes were cloned by limiting dilution. After 1 month in culture with T-cell growth factor several clones incorporated tritiated thymidine when stimulated with the appropriate allogeneic cells. Specificity of restimulation of two primed lymphocyte clones, designated 12-2 and 12-8, was studied in detail after varying periods of culture (up to 50 days). Clone 12-2 cells were stimulated only by cells expressing the HLA-Dw antigens of the original priming cells (Dw3); furthermore, this primed lymphocyte reagent specifically recognized antigens associated with only one of the three distinct Dw3-bearing haplotypes from an informative family (KOH). Clone 12-8 cells, on the other hand, failed to recognize Dw3 antigens in the random panel or on homozygous typing cells (including the original priming cell), but were strongly restimulated by certain cells expressing Dw4 antigens. In addition, within family KOH, these restimulating products segregated with another one of the three Dw3-bearing haplotypes but with none of the three Dw4-bearing haplotypes. These two clones exemplify a hitherto unknown precision in cellular typing of the HLA-D region. Clone 12-2 allows the discrimination of a probably rare and as yet undetected HLA-Dw3 subtypic specificity. Clone 12-8, on the other hand, apparently identifies an allelic system segregating with HLA but distinct from the HLA-D determinants definable by HTC-typing. [ABSTRACT FROM AUTHOR]

A panel of alloindifferent monoclonal antibodies (MAB's) was used in complement-dependent lysis to characterize human myeloid, erythroid and multipotential progenitors (CFU-GM, BFU-E, CFU-GEMM) for their expression of MHC class II HLA-DR, -DP, and -DQ products. 7-16 donors were tested in each system. MAB Tü 34, detecting DR products, caused reduction of CFU-GM by a mean of 89%, whereas BFU-E and CFU-GEMM were reduced by 67% and 66% respectively. 35% of CFU-GM, 27% of BFU-E and 32% of CFU-GEMM were lysed by MAB B7/21, recognizing HLA-DP determinants, while Tü 22, binding HLA-DQ antigens, lysed 32% only of CFU-GM and did not lyse the other progenitors. Employing the 'broad' MAB Tü 39, which binds at least DR and DP, inhibition of colony formation by CFU-GM was generally greater than that caused by Tü 34 alone or even by combinations of Tü 34, Tü 22, and B7/21. This suggests that there may be a subset of DR, DP, DQ hematopoietic progenitors, which nonetheless bind MAB Tü 39, previously proposed as a candidate for the recognition of novel class II antigens. [ABSTRACT FROM AUTHOR]

Regulatory effects of mixed lymphocyte culture (MLC)-derived CD4+ human T cell clones on granulocyte-macrophage colony (CFU-GM) formation by normal bone marrow (BM) were studied in an initial attempt to establish an in vitro model for the negative feedback control of myelopoiesis by alloactivated T cells. This is likely to be of clinical significance in the aberrant control of haematopoiesis during some cases of graft-versus-host disease (GVHD) after allogeneic BM transplantation. Whilst 5 such alloproliferative clones generally failed to suppress CFU-GM, the majority of clones with natural killer (NK)-like activity [10], or those with suppressive activity in MLC [2], regularly and strongly suppressed in this system, reinforcing the view that certain T cells may have potent negative regulatory effects on haematopoiesis. [ABSTRACT FROM AUTHOR]