Quantitative studies of the adoptive immunological memory in mice. II. Linear transmission of cellular memory.

A calibrated cell transfer system allows detection of the anamnestic response to albumin without interference from the host's immune machinery; it was used to study the immunological memory of mouse spleen cell populations. The secondary antibody-forming capacity of the transferred cells was measured by challenging them at periods up to 6 months after transfer. The peak levels attained show a declining pattern in two phases: during the first month with a half-life of 15 days; thereafter, with a half-life of 100 days. The corresponding half-lives of the cellular memory are 26 and 190 days. In the light of these and of radioinactivation data, immunological memory is defined as the persistence of a specifically determined stem cell line, along which the information necessary to give rise to an antibody-forming cell population is transmitted from mother to daughter cells.

Quantitative studies of the adoptive immunological memory in mice. I. An age-dependent barrier to syngeneic transplantation.

Antibody-forming cells suspended from a mouse spleen and transferred to intact animals of the same genotype face a barrier which severely affects their capacity to implant and/or to function. This phenomenon was quantitatively studied in a model system which, utilizing the immunogenic properties of human serum albumin in mice, allows the secondary response of the transferred cells to be followed without interference from the host's own reactivity. The barrier to syngeneic transplantation was found (a) to be radiosensitive (500 R X-rays to the recipient abolishes it and insures optimal functional conditions to the donor cells) in the same order of magnitude of other mammalian systems involving rapidly dividing cell populations, and (b) to depend upon the age of the recipient: its linear rise is documented from birth time (when approximately 50% of the maximal immune capacity of the transfer is expressed) to the age of 2 months ( approximately 1 %). The significance of these findings to the immune response and to cell growth and differentiation is discussed.

Antibody-mediated activation of a defective beta-D-galactosidase. II. Immunological relationship between the normal and the defective enzyme.

Two closely related protein antigens were used to study immunogenic competition. Namely, normal beta-D-galactosidase of Escherichia coli (Z) and a genetically defective beta-D-galactosidase (AMEF) which seems to differ from the normal in one amino acid substitution. A unique characteristic of this pair of antigens is that, although they are indistinguishable in precipitation and absorption tests with antibodies, the enzymatic activity of AMEF is specifically increased several-hundredfold in the presence of antibodies directed against Z. The following results show that Z and AMEF also differ in their immunogenic ability: (a) antibodies directed against Z activated AMEF; antibodies directed against AMEF did not activate, but competed specifically with activating antibodies. (b) Animals immunized with AMEF failed to produce activating antibodies when they were subsequently challenged with Z, although the presence of some cells primed to produce activating antibodies could be demonstrated by adoptive transfer. (c) Animals preimmunized with Z were stimulated in their production of activating antibodies by AMEF challenge, although not as efficiently as with Z. A model explaining these observations by competition for the immunogenic site among antigen-sensitive cells carrying cross-reacting receptors is presented.

Effect of antigen/antibody ratio on macrophage uptake, processing, and presentation to T cells of antigen complexed with polyclonal antibodies.

Activation of a galactosidase-specific murine T hybridoma clone and of a human tetanus toxoid-specific T clone by antigen-presenting cells (APC) was used to evaluate the regulatory function of antibodies complexed with the relevant antigen. Complexed antigen, in fact, is taken up with high efficiency thanks to Fc receptors borne by APC. Antibody/antigen ratio in the complexes proved to be a critical parameter in enhancing antigen presentation. Complexes in moderate antibody excess provided optimal T cell activation independently of the physical state of the complexes (precipitated by a second antibody or solubilized by complement). Complexes in extreme antibody excess, on the contrary, did not yield T cell activation although taken up by APC efficiently. The effect of antibodies at extreme excess was observed with substimulatory dose of antigen (loss of potentiation) and with optimal dose of antigen (loss of stimulation). An excess of specific polyclonal antibodies hampers proteolytic degradation of antigen in vitro, supporting the view that a similar mechanism may operate within the APC that have internalized immune complexes in extreme antibody excess. The possibility that immune complex forming in extreme antibody excess may turn off the T cell response is proposed as a regulatory mechanism.

Maturation of the immkune response in vitro. Focal fluctuation and changes in affinity of anti-beta-D-galactosidase activating antibody.

We have cultivated lymph node microfragments from beta-D-galactosidase (Escherichia coli) primed rabbits and have measured their secondary response directed towards the whole molecule (precipitating antibodies) and to a single determinant (activating antibodies) of the antigen. By decreasing the size of the fragments to 10(5) cells, we began to observe heterogeneity among identical cultures in terms of positivity of response, antibody specificity, and titers. The affinity of "early" activating antibodies was inversely proportional to the dose of challenge. While no maturation was seen in low and excessive challenge, in all cultures receiving intermediate doses the association constant was raised several orders of magnitude within periods of 20 days. The relevance of these data to the mechanism of affinity selection of antigen-sensitive cells is discussed.

Antibody raised against soluble CD4-rgp120 complex recognizes the CD4 moiety and blocks membrane fusion without inhibiting CD4-gp120 binding.

We studied the humoral response of mice immunized with soluble CD4-rgp120 complex, testing polyclonal and monoclonal antibodies (mAbs) with the aim of identifying molecular changes that take place after the first interaction between human immunodeficiency virus and the cell surface. The antisera had a paradoxically high syncytia-blocking titer associated with anti-CD4 specificity, while their capacity to inhibit CD4-gp120 binding was relatively modest. One of the mAbs produced from these responders blocks syncytia formation but does not inhibit CD4 interaction with gp120. Apparently, this mAb interacts with the CD4 moiety of CD4-gp120 complex and prevents a post-binding event necessary for membrane fusion and viral infection.

Recommendations of the Spanish brachytherapy group (GEB) of Spanish Society of Radiation Oncology (SEOR) and the Spanish Society of Medical Physics (SEFM) for high-dose rate (HDR) non melanoma skin cancer brachytherapy.

Clinical indications of brachytherapy in non-melanoma skin cancers, description of applicators and dosimetry recommendations are described based on the literature review, clinical practice and experience of Spanish Group of Brachytherapy and Spanish Society of Medical Physics reported in the XIV Annual Consensus Meeting on Non Melanoma Skin Cancer Brachytherapy held in Benidorm, Alicante (Spain) on October 21st, 2016. All the recommendations for which consensus was achieved are highlighted in blue. Regular and small surfaces may be treated with Leipzig, Valencia, flap applicators or electronic brachytherapy (EBT). For irregular surfaces, customized molds or interstitial implants should be employed. The dose is prescribed at a maximum depth of 3-4 mm of the clinical target volume/planning target volume (CTV/PTV) in all cases except in flaps or molds in which 5 mm is appropriate. Interstitial brachytherapy should be used for CTV/PTV >5 mm. Different total doses and fraction sizes are used with very similar clinical and toxicity results. Hypofractionation is very useful twice or 3 times a week, being comfortable for patients and practical for Radiotherapy Departments. In interstitial brachytherapy 2 fractions twice a day are applied.

Fetal dose measurements and shielding efficiency assessment in a custom setup of (192)Ir brachytherapy for a pregnant woman with breast cancer.

PURPOSE: To assess the radiation dose to the fetus of a pregnant patient undergoing high-dose-rate (HDR) (192)Ir interstitial breast brachytherapy, and to design a new patient setup and lead shielding technique that minimizes the fetal dose. METHODS: Radiochromic films were placed between the slices of an anthropomorphic phantom modeling the patient. The pregnant woman was seated in a chair with the breast over a table and inside a leaded box. Dose variation as a function of distance from the implant volume as well as dose homogeneity within a representative slice of the fetal position was evaluated without and with shielding. RESULTS: With shielding, the peripheral dose after a complete treatment ranged from 50 cGy at 5 cm from the caudal edge of the breast to <0.1 cGy at 30 cm. The shielding reduces absorbed dose by a factor of two near the breast and more than an order of magnitude beyond 20 cm. The dose is heterogeneous within a given axial plane, with variations from the central region within 50%. Interstitial HDR (192)Ir brachytherapy with breast shielding can be more advantageous than external-beam radiotherapy (EBRT) from a radiation protection point of view, as long as the distance to the uterine fundus is higher than about 10 cm. Furthermore, the weight of the shielding here proposed is notably lower than that needed in EBRT. CONCLUSIONS: Shielded breast brachytherapy may benefit pregnant patients needing localized radiotherapy, especially during the early gestational ages when the fetus is more sensitive to ionizing radiation.

An immuno-enzymatic assay of cortisol using E. coli beta-galactosidase as label.

An enzyme-assay of cortisol was established by using E. coli beta-galactosidase as a marker. The number of cortisol residues per molecule of enzyme and the method of separation of free from antibody-bound enzyme proved to be critical factors for the assessment of the assay. A sensitivity ranging from 100 to 150 pg of cortisol per tube has been achieved. A comparsion with other methods, i.e. competitive protein binding assay, radioimmunoassay and fluorimetry, is reported.

Use of bispecific hybrid antibodies for the development of a homogeneous enzyme immunoassay.

Hybrid bispecific monoclonal antibodies reacting with carcinoembryonal antigen (CEA) and with the E. coli enzyme beta-galactosidase (GZ) were produced by fusion of hybridomas or chemical linkage of half-antibodies. Since the original anti-GZ antibody used in these experiments was capable of protecting GZ from thermal denaturation, it was possible, by hybridizing it with two different non-competitive anti-CEA antibodies, to design a homogeneous enzyme immunoassay for quantitation of CEA. In fact, a mathematical analysis of the reaction indicates that, under appropriate concentrations of the reactants, circular complexes can be formed which contain the two hybrid antibodies, the GZ enzyme and the CEA antigen. The stability of these complexes can be expected to be substantially greater than that of the more labile CEA-free GZ-antibody complexes, prompting a significant increase in the amount of enzyme molecules which are bound to antibody and are consequently protected from thermal denaturation. These expectations were supported by experimental results: under appropriate conditions, heat-resistant enzyme activity was indeed proportional to concentration of CEA in the range up to 75 ng/ml. As predicted by theory, however, in the presence of excess CEA - in fact at CEA concentrations which are higher than those of possible clinical relevance - circular complexes tended to open up, leading to a marked prozone effect.

Methods for the selection and growth of antigen-specific cytolytic T lines and clones bearing a defined T cell receptor beta chain marker.

Murine cytolytic T lymphocyte (CTL) clones specific for type A influenza virus antigens were generated by in vitro stimulation with syngeneic virus-infected cells in the presence of T cell growth factor (TCGF). All CTL clones recognize viral determinants shared by PR8 and X31 influenza viruses in association with a class I antigen, coded either by the H-2K or H-2D end of the appropriate haplotype. All clones express the Lyt2 antigen marker. Two of five clones also express an antigenic determinant of the V beta chain of the T cell receptor (TCR) identified by F23.1 monoclonal antibody. To effectively generate F23.1+ and antigen-specific CTL clones, heterogenous CTL lines were expanded with F23.1 coated Sepharose beads in the presence of TCGF and then stimulated with PR8 virus-infected cells. Thus, both the proliferative activity to PR8 and the expression of the F23.1 marker was increased significantly. Alternatively, F23.1+ T cells were sorted from in vivo primed mice and expanded with PR8 virus-infected stimulator cells in the presence of TCFG. This F23.1+ T cell line exhibited antigen-specific cytotoxicity for PR8 virus-infected target cells. Additionally, in an 'FcR-focused killing' assay only the F23.1+ CTL line and F23.1+ clones lysed Fc receptor bearing target cells in the presence of F23.1 antibody. These findings indicate that antigen-specific and F23.1+ clones can be selected with high efficiency by alternating stimulation with influenza virus-infected cells and with F23.1-coated Sepharose beads or through the use of a cytofluorograph. The usefulness of antigen-specific and F23.1+ CTL clones and other possible strategies for their selection are discussed.

An enzymatically active antigen-antibody probe to measure circulating immune complexes. II. E. coli beta-galactosidase in the probe and C1q as the recognition unit.

An enzymatically active probe (beta-galactosidase-anti-beta-galactosidase complex) is used to measure circulating immune complexes (CIC), in a competition assay where probe and CIC are confronted with a 'recognition unit'. The latter is bovine conglutinin in the original description of this method. Here we describe a version utilizing human or bovine C1q. The two techniques are compared for their sensitivity and specificity, on both in vitro formed tetanus toxoid-anti-toxoid complexes and on sera from patients with selected diseases. The results confirm that the two recognition units are sensitive to families of CIC that only partially overlap. The parallel use of conglutinin and C1q yields both quantitative and qualitative information on the nature of CIC in individual sera.

The serum capacity to solubilize immune complexes (ICSC) measured by an enzyme-anti-enzyme complex probe.

The capacity to solubilize immune complexes can be readily measured by incubating the test serum with a suspension of an immune precipitate formed by beta-galactosidase and anti-beta-galactosidase antibody, and then reading the enzyme units (EU) liberated in the clear supernatant. Our method is rapid and inexpensive; it can be performed in plates and read in scanning colorimeters. Although on large numbers of observations the ICSC is significantly correlated with the CH50, a few discordant cases suggest that solubilization and haemolysis are functions of the alternative and classical pathways of complement respectively.

Inhibition of the accessory function of murine macrophages in vitro by cyclosporine.

The accessory function of macrophages, which is strictly related to the induction of T cell activation, has been studied to determine whether it is affected by cyclosporine (CsA). Irradiated spleen cells, used as a source of macrophages, were pulsed overnight with beta-galactosidase (GZ) in the presence of CsA. After washing of the pulsed macrophages, cells from a GZ-specific T cell line were added to cultures and 3H-thymidine incorporation was measured 72 hr later. We found that 500 ng/ml CsA present during macrophage pulsing with GZ reduced T cell proliferation to 5%. On the other hand, 100 ng/ml CsA almost completely abrogated the proliferative response when present for the duration of the culture. Similar results were also obtained using antigen-pulsed peritoneal-adherent macrophages to stimulate the T cell line to proliferate, or a T hybridoma clone to produce interleukin-2 (IL-2). The possibility that CsA actually affects interleukin-1 (IL-1) production by macrophages by inhibiting uninvolved T cells could be ruled out. We conclude that CsA-induced inhibition of T cell functions (proliferation and IL-2 production) is partially due to the effect of the drug on the accessory function of macrophages. This immunosuppressive mechanism of action of CsA on macrophages has not been previously described.

Effect of cyclosporine on the antigen-presenting function of human and murine accessory cells.

Recent reports have challenged the belief that accessory cells are resistant to cyclosporine. Such a tenet was based on the observation that several functions of accessory cells, such as IL-1 production and phagocytosis, are resistant to the drug. On the other hand, when a less primitive, more refined function of accessory cells was examined--i.e., the capacity to take up, process, and present antigen in an MHC-restricted fashion to antigen-specific T lymphocytes, CsA proved to be an effective inhibitor. In contrast to this finding, when antigen was provided in the form of an immune complex prepared with a monoclonal antibody, uptake of antigen--likely mediated by the Fc receptors--and subsequent processing and presentation were not affected by CsA. These results suggest that, depending on whether the antigen is taken up by constitutive or by receptor-mediated endocytosis, accessory cells can be functionally defined as resistant or sensitive to CsA.

Difference in sensitivity to cyclosporine in vitro of human alloreactive lines and clones.

The effectiveness of cyclosporine (CsA) as immunosuppressive agent in human kidney graft rejection is well established. However, in spite of efforts to maintain optimal plasma levels, a fraction of transplanted patients undergo rejection episodes and/or irreversible chronic rejection. This suggests that immunosuppression by CsA cannot control the alloreactive response if there is a high degree of histoincompatibility for HLA or non-HLA antigens, or it has little effect on the "high responder" patient. Both possibilities are difficult to test in the human system. A third hypothesis, the existence of individual CsA resistance, was tested by evaluating the in vitro inhibitory activity of CsA on alloreactive T cell lines from several individuals. A different degree of in vitro sensitivity to the drug was observed among alloreactive lines generated from different individuals and among clones obtained from the same bulk line. The variability at the individual level and at the clonal level may account for the onset of CsA-resistant rejection assuming that in vivo a positive selection in the presence of the drug occurs and allows for the resistant clones, if present, to dominate the sensitive ones.

Recognition of donor fibroblast antigens by lymphocytes homing in the human grafted kidney.

A human transplanted kidney, surgically removed because of untreatable chronic rejection, was used as the source of lymphocytes (K-L) of recipient origin that were expanded with interleukin-2 (IL-2), and of kidney fibroblasts (K-F) of donor origin that were maintained as an established line. Cytotoxicity assays were performed using K-L and peripheral blood lymphocytes (PBL) as effectors, and K-F and donor PBL as targets. From the results the following conclusions can be drawn: (1) cytotoxic lymphocytes, presumably involved in the process of chronic graft rejection, home in the kidney (from which they can be recovered) but are not detected in the circulation; (2) cytotoxic lymphocytes can be generated from peripheral lymphocytes by mixed lymphocyte culture (MLC) and further expansion in vitro with IL-2 (MLC-L); and (3) although both K-L and MLC-L are cytotoxic toward K-F, the former are not cytotoxic toward donor PBL. This suggests that although MLC-L recognize antigens shared by K-F and PBL, K-L recognize antigens specific for K-F only. These results, if confirmed, indicate that antigens not present on PBL, and possibly tissue-restricted are important in graft rejection. Thus, while monitoring transplanted patients, a lack of cytotoxicity in the recipient PBL may be misleading because the relevant cytotoxic effector cells may have disappeared from the periphery and the appropriate antigenic target may be absent on donor PBL.

Autoantibodies to CD4 in HIV type 1-exposed seronegative individuals.

The aim of this study was to investigate the presence and the fine specificity of anti-CD4 autoantibodies in seronegative subjects sexually exposed to HIV-1. Anti-CD4 autoantibodies were previously detected in a fraction of HIV-1-seropositive individuals. Whole sera, purified IgG fractions, and supernatants of EBV-transformed lymphoblastoid cell lines were analyzed by means of ELISA, Western blot, and by competition assays using monoclonal antibodies with known fine specificities. Anti-CD4 antibodies were found in 6 of 18 individuals exposed to HIV-1 infection and who have been persistently seronegative. These antibodies inhibited HIV-1-driven syncytium formation, did not interfere with the CD4-gp120 interaction, and competed for CD4 binding with two of three anti-CD4 monoclonals with known fine specificities. Moreover, autoantibodies with the same fine specificities were found in the supernatants of oligoclonal EBV-transformed B cell lines derived from these individuals. At variance, in the HIV-1-positive patients included in our study, the anti-CD4 antibody response was directed to a broader panel of epitopes, including those involved in CD4-gp120 interactions. In conclusion, anti-CD4 antibodies specific for defined epitopes of the CD4 molecule are generated in the course of an early immune response to HIV-1 antigens in the absence of other signs of infection, as they can be detected by conventional methods. These autoantibodies may play a protective role either alone or in association with other cellular and humoral factors.