Soluble interleukin-2 receptors and autoantibodies in the serum of healthy elderly individuals

Recently, we reported an increased incidence of various autoantibodies in a healthy elderly population (Group A, 64 subjects). Presently we examined whether there is variability in the expression of the age-associated immunological aberrations between different geriatric populations by extending our observations in another healthy elderly population (Group B, 119 subjects). We also determined the serum levels of soluble IL-2 receptors (sIL-2R) attempting to define the activation status of the immune system during senescence. Compared to non-elderly controls, healthy elderly individuals exhibited a significantly higher incidence of autoantibodies as well as significantly higher levels of sIL-2R in serum (p less than 0.001), the latter possibly suggesting the occurrence of lymphocytic activation during the ageing process. The overall prevalence of autoantibodies was statistically associated with the presence of raised sIL-2R levels in serum (p less than 0.005). These aberrant immunological phenomena were more frequent among the elderly of group A, compared to group B (p less than 0.005). In contrast to the uniform expression of various autoantibodies previously observed in group A, the autoantibody profile of group B consisted mainly of rheumatoid factor and antibodies to single-stranded DNA. Finally, no association could be demonstrated between the presence of autoantibodies and HLA antigens in 42 elderly studied.     

Anti-Idiotypes to HLA and their Role in Transplantation

The network theory proposed by Jerne is based on the finding that variable regions of T and B cell antigen receptors are structurally diverse and express unique variable region determinants. We have postulated that idiotypes present on the V region of the anti-HLA antibody molecule (Ab1) can elicit the production of anti-anti-HLA antibodies or Ab2 and that such Ab2 may play a role in the suppression of anti-HLA antibody responses. We first tested the validity of this concept in pregnancy, natures most perfect model of allogeneic tolerance. Our studies revealed that anti-Id antibodies were present during pregnancy at times when HLA antibodies had disappeared from the circulation (1,2). This inverse correlation between Ab1 and Ab2 suggested that Ab2 suppresses the production of Ab1. Once the validity of this concept was substantiated in the model of pregnancy, we tried to determine whether anti-Id antibodies to HLA also play a role in the down regulation of the alloimmune response to transfusions and transplantation. For this, we monitored patients for the development of anti-anti-HLA antibodies following donor specific blood transfusion (3). We found that most patients developed Ab2 to the mismatched HLA class II antigens of the blood donor two weeks following transfusion and were associated with successful transplantation. Furthermore, we found a positive correlation between the presence of anti-anti-HLA antibodies and tolerance to the graft in patients with a history of presensitization to HLA antigens of the donor (4). The role of anti-Id antibodies to HLA was also evaluated in renal and heart transplantation (5,6,7). In these studies, patients were monitored following transplantation for the production of Ab2. We found that 5 year actuarial survival of heart and kidney allografts was significantly higher in patients developing Ab2, compared to patients without Ab2. Taken together, our data suggest that anti-Id antibodies to HLA play an important role in suppressing the immune response to HLA and that such anti-Id may be of therapeutic interest in transplantation.     

Anti-Idiotypes to HLA and their Role in Transplantation

The network theory proposed by Jerne is based on the finding that variable regions of T and B cell antigen receptors are structurally diverse and express unique variable region determinants. We have postulated that idiotypes present on the V region of the anti-HLA antibody molecule (Ab1) can elicit the production of anti-anti-HLA antibodies or Ab2 and that such Ab2 may play a role in the suppression of anti-HLA antibody responses. We first tested the validity of this concept in pregnancy, natures most perfect model of allogeneic tolerance. Our studies revealed that anti-Id antibodies were present during pregnancy at times when HLA antibodies had disappeared from the circulation (1,2). This inverse correlation between Ab1 and Ab2 suggested that Ab2 suppresses the production of Ab1. Once the validity of this concept was substantiated in the model of pregnancy, we tried to determine whether anti-Id antibodies to HLA also play a role in the down regulation of the alloimmune response to transfusions and transplantation. For this, we monitored patients for the development of anti-anti-HLA antibodies following donor specific blood transfusion (3). We found that most patients developed Ab2 to the mismatched HLA class II antigens of the blood donor two weeks following transfusion and were associated with successful transplantation. Furthermore, we found a positive correlation between the presence of anti-anti-HLA antibodies and tolerance to the graft in patients with a history of presensitization to HLA antigens of the donor (4). The role of anti-Id antibodies to HLA was also evaluated in renal and heart transplantation (5,6,7). In these studies, patients were monitored following transplantation for the production of Ab2. We found that 5 year actuarial survival of heart and kidney allografts was significantly higher in patients developing Ab2, compared to patients without Ab2. Taken together, our data suggest that anti-Id antibodies to HLA play an important role in suppressing the immune response to HLA and that such anti-Id may be of therapeutic interest in transplantation.     

Donor-specific sensitization by cadaveric venous allografts used for arterial reconstruction in peripheral arterial occlusive vascular disease

The use of allogeneic venous grafts from postmortal organ donors allows for the reconstruction of critically affected arteries in patients with peripheral occlusive vascular disease. We were interested to determine the prevalence and specificity of anti-HLA antibodies in patients after allogeneic vein transplantation. Anti-HLA class I and II alloantibodies were analyzed by flowcytometric analysis using color-coded microbeads coated with HLA antigens including recombinant single antigens. Nine out of 10 patients involving 12 venous allografts were positive for anti-HLA alloantibodies. All antibody-positive patients carried both anti-HLA class I and II alloantibodies. Anti-donor HLA specificity of the anti-HLA alloantibodies was seen in seven out of nine patients for anti-class I antibodies and in eight out of nine patients for anti-HLA class II antibodies. A high rate of donor-specific allosensitization was seen after allogeneic venous transplantation. In conclusion, allosensitization not only includes a humoral response against the constitutively expressed class I antigens but also extends to class II antigens.     

Perturbation of the immune network in herpes gestationis

We have studied the development of anti-idiotypic antibodies to HLA and of autoantibodies reacting with alloactivated T lymphoblasts in a woman with herpes gestationis (HG). This primigravida developed an elevated titer of anti-HLA antibodies, (Ab1), in association with a low titer and late appearance of anti-anti-HLA antibodies (Ab2). At delivery she developed only minimal levels of antibodies reacting with autologous T lymphoblasts (T1), sensitized against the immunizing HLA antigens of the child. Her serum reacted, however, with T lymphoblasts, primed in AMLC against autologous T blasts alloactivated in vitro against her husband (T2). Because healthy gravidae do not show such antibodies, it appears that they are peculiar to and may represent a perturbation of the idiotypic network in regard to the immune     

Cell culture density modulates the incorporation of HLA antigens by enveloped viruses

Uninfected, as well as feline leukemia virus (FeLV) infected human cells cultured under high cell density conditions undergo changes in the expression of major histocompatibility complex (MHC) antigens, as determined by indirect trace binding radio immunoassay (RIA) using monoclonal anti-HLA antibodies and by decreased sensitivity to complement mediated cytotoxicity by anti-HLA alloantibodies. FeLV particles produced by the viral infected cells are also sensitive to neutralization by anti-HLA antibodies, suggesting that enveloped viral particles incorporated MHC antigens in the viral envelope. The amount of HLA antigens expressed in the viral enveloped, closely reflects the expression of HLA antigens by the virus-producer lymphoid cells. FeLV-infected HsB-2 (T) and SB (B) lymphoid cells cultured under high cell concentration condition show decreased expression of some HLA antigens (A2, B12, B17), and the viral particles produced by those cells also incorporate lower amounts of such antigens. Our results, based on the findings that human lymphoid cells (uninfected, as well as FeLV infected) show decreased expression of some HLA membrane determinants when growth under high cell density conditions, indicate that no viral selective mechanism operates in the incorporation of HLA determinants by enveloped viruses. Instead, our results suggest that viruses pick up MHC antigens from the host cell membrane according to the concentration of those antigens on the surface of the cells at the time of virus budding.     

Expression of HLA antigens on leukaemia cells

Virus infected cells can carry HLA antigens not demonstrated in uninfected cells. In a wider context, it is known that tumour cells in the mouse can exhibit H2-like antigens of foreign haplotypes which they resemble. Our aim was to investigate, using an absorption method, the HLA antigens of ten leukaemic cells and to compare these results with those of normal lymphocytes. The following sera was used for absorption: anti-B5 serum, anti-B7 serum, anti-B12 serum. These sera were absorbed on the ten leukaemic cells and fifteen healthy lymphocytes. An anti-A1 + B8 serum and an anti-A2 serum were absorbed on the ten leukaemic cells, but there was not enough serum to carry out the similar absorption with the healty lymphocytes. Two points emerged from these results. (1) Leukaemia cells absorbed anti-HLA antibodies as effectively as healthy lymphocytes when the cells carried the antigen corresponding to the serum specificity. The curves obtained with leukaemia cells were comparable to those of healthy cells. (2) When the cells were not carriers of the antigens corresponding to the antibody's specificity, leukaemia cells were also capable of absorbing antibodies, unlike healthy lymphocytes which had no or poor absorption.     

The Influence of Intravenous Immunoglobulin Treatment on Maternal Immunity in Women With Unexplained Recurrent Miscarriage

PROBLEM: Recently the protective value of high-dose intravenous immunoglobulin (IVIG) in the treatment of unexplained recurrent miscarriage has been reported to be similar to that of conventional immunotherapy with paternal leukocytes. We examined the effect of IVIG treatment on the cellular and humoral level of maternal immunity to demonstrate the possible mechanism by which IVIG might act to prevent recurrence of pregnancy loss. METHOD: Eight patients were treated with a 20- to 25-g dose of IVIG every 2 to 3 wk during their first-trimester pregnancies. The development of anti-idiotypic autoantibodies against maternal T-cell receptors, maternal anti-paternal lymphocyte antibodies detected by flow cytometric crossmatch, and changes of maternal lymphocyte subpopulations were monitored before pregnancy and then weekly during IVIG treatment. RESULTS: Five of eight patients gave birth successfully after IVIG treatment given during the first trimester of pregnancy (success rate: 62.5%). Although we could not demonstrate a general immunological effect of IVIG on maternal immunity in vivo, a few significant changes of immunological parameters were found in some patients. CONCLUSION: Our results suggest that the effect of IVIG on maternal immunity is not a passive increase of blocking antibody including anti-HLA antibody or modification of maternal T-cell subsets but, more likely, a passive increase of anti-idiotypic antibody against anti-HLA antibody or soluble HLA antigens. However, whether the immunomodulating effect of IVIG is related to its possible mechanism to prevent abortion remains unestablished.     

Humoral immunity in hand transplantation: Anti-HLA and non-HLA response

Antibodies against donor’s HLA antigens and B cell activity are recognized modulators of immune response to allograft. The role of both anti-HLA and non-HLA antibodies is understood in solid organ transplantation, but has not been addressed in composite tissue allografts.     

Immunological features of juvenile onset diabetic patients correlated to HLA type.

Ninety-six juvenile onset type diabetics showed an increase in the frequency of HLA B8 and B15 and a decrease in frequency of HLA B7 antigens. Sixty-four maturity onset diabetics showed no disturbance in the frequency of these antigens. Fifty-four of the juvenile onset type diabetics, with an average duration of disease of 3.2 years were tested for the presence of islet cell antibodies (ICAs). Thirty-two per cent were positive, the incidence decreasing from 70% in those patients tested within 1 year of diagnosis to zero in those patients tested within 1 year of diagnosis to zero in those patients tested more than 5 years after diagnosis. No correlation was found between the incidence of ICAs and either cell-mediated immune reactions or HLA type. B15 positive patients were associated with cell-mediated immune reactions to pancreatic antigens and with the presence of other tissue autoantibodies. HLA phenotypes were not associated with environmental data. Diabetic siblings had identical HLA A-B haplotypes more often than could be expected to occur by chance.     

Class I antigens of the major histocompatibility complex on cytotrophoblast of human chorion laeve

Twenty amniochorions from normal, term pregnancies were studied immunohistologically with the use of well-characterized monoclonal antibodies to beta-2-microglobulin (beta 2M) and to common determinant of HLA-A, -B, -C. In the same study, polyclonal antiserum to trophoblast antigens (TA) were employed with double fluorochrome labels to determine if cytotrophoblast in the chorion laeve expressed TA, HLA or both. The results showed that, in the majority of cases, HLA was not identified on trophoblast, but TA were. However, some cytotrophoblast within the amniochorionic mantle were non-reactive with anti-TA serum and were positive with monoclonal antibodies to HLA and beta 2M antigens. Since these structures were identified as cytotrophoblast by a battery of techniques, in this report they are tentatively designated as metatrophoblast. The role of these cells in the materno-trophoblastic relationship in normal human pregnancy has yet to be determined. This is not envisaged as a simple matter, for metatrophoblast can be recognized by anti-beta 2M and anti-HLA (W6/32), but not by anti-HLA (61D2), suggesting that their expression of class I histocompatibility complex (MHC) antigens may be incomplete, or that there may be a contribution of antigens from an extra-embryonic MHC.     

Signal transduction in lymphocyte activation through crosslinking of HLA class I molecules

The inhibitory effect of anti-HLA class I monoclonal antibodies on lymphocyte proliferation has been well documented. However, recent data suggest that anti-HLA class I monoclonal antibodies can enhance lymphocyte proliferation via both anti-CD3-induced {1,2} and anti-CD2-induced {3} activation pathways. Here we demonstrate that both inhibition and activation can be regulated by the degree of aggregation of HLA class I antigens. Crosslinking of monoclonal antibodies specific for HLA-A, HLA-B, or monomorphic determinants (using anti-IgG2 and/or anti-Ig “second step” monoclonal antibodies) increased the capacity of the anti-HLA class I monoclonal antibodies to inhibit phytohemagglutinin-induced proliferation. However, the cytosolic free calcium concentration was increased in CD4+ cells, CD8+ cells, B cells, and CD16+ cells when anti-HLA class I monoclonal antibodies were crosslinked, suggesting that an activation signal was generated by aggregation of the corresponding antigens. Indeed, inositol 1,4,5-trisphosphate could be detected in peripheral blood lymphocytes following crosslinking of anti-HLA class I monoclonal antibodies. Class I aggregation also induced proliferation of peripheral blood mononuclear cells in the presence of submitogenic doses of phorbol 12-myristate 13-acetate. Strong conditions of crosslinking (monomorphic monoclonal antibody plus both anti-IgG2 and anti-Ig ) induced CD25 expression and responsiveness to recombinant interleukin 2. Our results suggest that aggregation of HLA class I antigens primed cells to become activated in the presence of progression signals including phorbol 12-myristate 13-acetate, recombinant interleukin 2, or anti-CD5 plus anti-CD28 monoclonal antibodies.     

Xenogeneic recognition of soluble and cell surface HLA class II antigens by proliferative murine T cells

In order to characterize the murine anti-human xenogeneic mixed lymphocyte reactions (MLR), we studied T cell proliferative responses against various human lymphoid cells by immunization of mice either with cellular or purified HLA-DR antigens. Data presented here indicated that small amounts of soluble HLA-DR antigen were able to prime mice, and that the xenogeneic MLR depends on the expression of HLA class II antigens on the stimulating cells. Experiments using a mutant cell line clearly showed that HLA-DP molecules were also sufficient in eliciting a primary or a secondary xenogeneic MLR while no secondary proliferative response was obtained with cells expressing only HLA class I molecules. Using a large panel of human cells with various haplotypes, our results also showed that (a) nonpolymorphic determinants of HLA class II antigens trigger dominantly the murine T cells and (b) the xenogeneic response required I-E and L3T4 accessory molecules and was not inhibited with anti I-A and monomorphic anti-HLA class II antigen monoclonal antibodies. Altogether these results suggest that HLA class II antigens act as nominal antigens in triggering a murine anti-human proliferative response.     

Increased Frequency of HLA-DR/v3 in Systemic Lupus Erythematosus

The distribution of HLA antigens was studied in 40 patients suffering from systemic lupus erythematosus. For loci A and B, increased frequencies of A1, B8 and the presence of only one B antigen were found. Nevertheless, in relation to the number of antigens tested, this increase was not significant. For the DRw locus, DRw3 was significantly increased (P < 0.001, after correction for the 39 antigens tested). However, the patients with DRw3 did not show any correlation with a specific clinical picture or the presence in their serum of lymphocytotoxic antibodies or autoantibodies against T and B lymphocytes.     

Good kidney transplant outcome in recipients with presensitization against HLA class II but not HLA class I

It is a matter of debate whether pretransplant anti-human leukocyte antigen (HLA) class II antibodies contribute to the increased graft rejection rate found in presensitized recipients. We investigated the influence of preformed anti-HLA class II antibodies on graft survival in 5949 cadaver kidney transplants. Pretransplant recipient sera were tested in enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (Ig)G-anti-HLA class I and IgG-anti-HLA class II antibodies. A total of 672 recipients with antibodies against HLA class II but not against HLA class I had a 3-year graft survival rate of 80 +/- 2%, identical to the 80 +/- 1% rate in 4561 recipients who were negative for anti-HLA class I and II (p = NS). Graft survival was significantly lower in 365 recipients who were positive for both anti-HLA class I and II (65 +/- 3%, p < 0.0001). Compatibility for HLA-A+B+DR influenced graft survival significantly in anti-HLA class I- and II-positive recipients (p = 0.0016), whereas no significant HLA effect was found in patients with antibodies directed against only class I or II. Surprisingly, not even incompatibility for HLA class II antigens of the DR locus caused a significant impairment of graft survival in anti-class II-positive recipients. We conclude that the risk associated with sensitization against HLA class II in the absence of sensitization against HLA class I is negligible.     

Nonrandom association of cellular antigens with HTLV-III virions

Retroviruses are known to incorporate cellular antigens as they bud from infected cells. To identify the cellular antigens that associate with the AIDS-retrovirus, we evaluated a preparation of HTLV-III antigens with a panel of monoclonal antibodies reactive with a variety of antigens expressed on the H9 T-cell line used to produce the virus. Only monoclonal antibodies that identified HLA class-II antigens, beta-2 microglobulin, and a single anti-HLA class-I antibody were reactive in an ELISA of solubilized HTLV-III virus. No reactivity was seen with 11 monoclonal antibodies to T-cell antigens or with five antibodies to determinants on HLA class-I A or B molecules. These data suggest that on H9 cells the association of budding HTLV-III virions with cellular antigens may be a nonrandom process in which some HLA antigens, particularly class-II antigens, are selectively incorporated into the viral envelope. It is possible that a selective association of HLA class II antigens with budding HTLV-III virions may also occur for T cells infected in vivo, and could have relevance for the pathogenesis of this virus.     

HLA antigens expressed on human ervthroid burst forming cells

The human major histocompatibility antigens HLA, play an important role in transplantation. To ascertain the expression of the antigens of A and B loci on the surface of human hemopoietic progenitors, the cytotoxic effect of specific anti-HLA sera was examined. Anti-HLA A2 and B5 sera were used in the present experiments. Circulating blood mononuclear cells and nucleated marrow cells, pre-treated with appropriate specific anti-HLA sera and complement, formed fewer colonies from BFU-e and CFU-c in a culture medium than the controls which were treated with autologous serum. In our experiments, the HLA antisera also killed macrophages, monocytes and T cells which carry the surface antigen. Then, the influence of macrophages, monocytes, and T cells on colony formation from BFU-e and CFU-c was examined. These results indicated that BFU-e as well as CFU-c expressed HLA antigens on their surface.     

Production in vitro of antibodies directed against alloantigen-specific recognition sites on T cells and on lymphocytotoxic HLA antibodies.

We have examined the mechanism of immunological unresponsiveness in a recipient (P.S.) with a long-term functioning renal allograft. P.S., whose HLA type is A1, A30; B14, B18; DR1, w8; DRw52; DQw1 and in whose serum we had earlier demonstrated the presence of antiidiotypic antibodies, received a kidney from a cadaver donor of HLA type A1, A10, B8 in March, 1970. Peripheral blood B lymphocytes from the patient were transformed with Epstein-Barr virus (EBV), and by the cluster-picking technique a B cell line was propagated with continuous production of antibodies. Antiidiotypic antibodies with two distinct biological functions were demonstrable; one specifically inhibiting the lymphocytotoxic activity of anti-HLA-B8, B5, and DR3 reference typing sera, and the other specifically inhibiting proliferative responses in MLC of the recipient's lymphocytes and of third party cells sharing B14, DR1, DQw1 with the patient against stimulator cells carrying B8, DR3 antigens. Immunodepletion experiments demonstrated that the inhibitory activity was associated with the IgM fraction. Absorption experiments suggested that different antibodies may be responsible for the inhibition of lymphocytotoxic activity of anti-HLA sera and of the proliferative responses in MLC. Antiidiotypic antibodies have been postulated to be important in maintaining allograft tolerance in vivo, thereby enhancing renal allograft survival. The availability of such antibodies in large quantities, produced in vitro, could provide antisera for the immunochemical characterization of specific idiotypic receptors on immunoglobulins and T lymphocytes.     

The pathogenesis of HLA-associated psoriasis

Genes for HLA antigens Bw17 and B13 are associated with psoriasis in about half of all cases of the disease. Differences between HLA - Bw17 and B13 associated psoriasis warrant separate designation of these two forms in studies of the disease and its treatment. Frequency of the genes and of psoriasis may be maintained by hypothetical resistance to specific infectious diseases. Since most of the functions regulated by genes known to be closely linked to HLA genes are immunologic, it may be hypothesized that psoriasis is immunologically determined. There is no evidence that psoriasis is caused by cutaneous infection. Cross-reactions between streptococcal and HLA antigens are not HLA type-specific. There is no direct evidence that circulating immune-complexes cause psoriasis, but HLA-B13 psoriasis is frequently preceded by streptococcal upper respiratory infection. The presence of immune-complexes containing streptococcal antigens should be looked for in the serum and skin lesions of B13 psoriasis. Although stratum corneum autoantibodies are found in normal serum, titers should be determined in psoriatics of known HLA types and correlated with localization of immuno-globulin in the stratum corneum in lesions. The possibility that keratinocytes bearing particular HLA antigens may be complement receptors should be explored.     

The Significance of Natural Autoantibodies

Since Burnet first introduced his “forbidden clones” theory, the discrimination between self and non-self and the physiologic mechanisms of avoiding autoimmunity remained an enigma. The realization in the past two decades that autoantibodies reacting with various self antigens are common in normals has led to intensive research on the origin and physiologic role of these “natural autoantibodies”. After reviewing the extensive literature on the appearance of natural autoantibodies in normal animals and humans, and the studies proving unequivocally that natural autoantibodies are coded by gern line genes, we will discuss the current hypotheses explaining their appearance and physiologic role. Despite the fact that numerous hypotheses explaining the origin of natural autoantibodies have been postulated only the two important ones will be discussed. The first, proposed by Cunningham, holds that clonal deletion as viewed by Burnet operates in early life; however, later in life all autoreactive B cells not eliminated, during ontogeny are prevented from expanding and secreting anti-self antibodies by a compensatory suppressor mechanism. Therefore, natural autoantibodies are postulated to be autoantibodies which are produced only in minute quantities allowed by the suppressor mechanism. The second hypothesis views autoantibody formation as a result of cross reaction between foreign and self determinants. It is suggested that the part of the B cell population which gives rise to autoantibodies carries a polyspecific receptor; fixation of a foreign antigen to this receptor induces the B cell to undergo a series of divisions and mutations, which under the selective pressure of the antigen leads to production of a highly specific antibody. Thus natural autoantibodies may constitute the antibodies secreted by these B cells prior to encountering foreign antigens. The biologic role of natural autoantibodies is also elusive. The common denominator to all the theories dealing with that puzzling question is the view that natural autoantibodies have a positive role in normal immune reactions, perhaps even an essential role without which normal immune function would be disrupted. Grabar suggested that natural autoantibodies are part of a physiologic mechsnism for cleansing the organism of self and non-self products in which classical antibodies serve to clear the body of foreign invading agents, while natural autoantibodies rid the organism of its own catabolic products. Cohen and Cooke suggested that natural autoantibodies, by binding to self antigens, act as a filter preventing powerful immune response against self triggered by self mimicking determinants on foreign invading microorganisms. Others have suggested a role for natural autoantibodies in the idiotypic network. We propose a different function for natural autoantibodies, namely enhancing host immune reactions to foreign infectious agents, in a similar manner in which HLA antigens participate in the activation of B and T cells, The question of the origin and biologic role of natural autoantibodies is not a purely academic one, and understanding these mechanisms will certainly clarify the pathogenesis of autoimmune diseases and autoimmunity in general.