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This new journal, Medical Immunology, was conceived to fill the large divide between the science of basic immunology and the application of this scientific knowledge to the diagnosis, treatment and prevention of diseases of the immune system. Why is there a gap between the basic science of immunology and the application of this science to medicine? The answers to this question are historical in large part, in that the immune system has only become recognized and defined within the last century. Moreover, the rate of accumulation of new information has accelerated markedly, with most of our present understanding acquired only within the past 20 years. Consequently, most of what we know about the immune system has only come into being within the professional lifetimes of the practicing medical community. Because of this recent explosion in knowledge, there simply has not been enough time for the translation to clinical medicine to occur. There is a large community of basic scientists who have been educated in the last few decades and who have contributed to the current state of knowledge in immunology. By comparison, because of "turf" concerns amongst faculties of medical schools, immunology course time in medical schools has been miniscule if covered in the curriculum at all. Consequently, most practitioners have not received much formal education in immunology. My medical colleagues often tell me that the immunological acronyms and jargon are huge and obfuscating, and they have no idea what the immunologists are talking about. There is definitely a gap between the immunologists and the clinicians. Even so, because of the rapid advances that have been made, especially within the last 20 years, we now have a new understanding of how the immune system functions, and we have available to us many new molecules to use as therapeutics. As well, because of advances in molecular genetics, we now have the capacity to engineer new molecules and vaccines at will. Consequently, there is now a tremendous need for clinical research to determine how best to use these new therapeutics, particularly, without toxicity. Because the modern science of immunology has only sprouted within the last 2–3 decades, most of those who contributed to our present view of the immune system are alive and well and still contributing. Therefore, by taking an historical view on this newest "organ system", it is easiest to gain an appreciation of what we now know, by focusing on those who have made a significant impact on medical immunology. As well, such a review should serve to illuminate those areas that promise to be the most fruitful areas for future development.

There is now effective therapy for infection by the Human Immunodeficiency Virus (HIV), but there is no cure. Consequently, antiviral drugs must be administered continuously to suppress viral replication. Recently, a large phase III international immune-based therapy trial was discontinued because it is difficult to measure clinical endpoints while antivirals are administered. Since the immune system has evolved under the selective force of microbial infections, the immune reaction is antiviral. This commentary explores the rationale of using "Diagnostic Treatment Interruptions" of antiviral therapies to determine efficacies of immune-based therapies.

Measurement of glucocorticoid receptors appears to be useful for selecting which patients with leukemia and lymphoma should receive glucocorticoid therapy. To determine the effect of recent or concurrent glucocorticoid therapy on the number of measured tumor glucocorticoid receptor sites, 18 patients with leukemia and lymphoma were studied. Baseline determinations of numbers of glucocorticoid receptors were performed on the malignant cells circulating in the patients' peripheral blood. Glucocorticoid therapy was then instituted consisting of dexamethasone 4 mg p.o. every 6 hr. Repeat determinations of the number of glucocorticoid receptor sites were performed within 24 hr and at various subsequent times from the start of therapy. When compared to baseline receptor numbers, 16 of 18 patients demonstrated a decrease in receptor number (median decrease 1651 sites/cell) after the start of glucocorticoid therapy. The magnitude of the change in receptor number was independent on the initial number of receptors. Our results suggest that in order accurately interpret glucocorticoid receptor numbers in patients with leukemia and lymphoma, glucocorticoid should not be administered for 3 wk prior to determinations of receptor levels.

Glucocortoid receptor (GR) levels have not been extensively studied in untreated patients with chronic lymphocytic leukemia (CLL). We have measured tumor GR levels using a whole cell assay in 18 untreated patients with CLL and correlated these levels with various in-vitro and in-vivo data. In 12 of these patients, tumor cells from more than one tissue were simultaneously studied. Measurable GR levels were found in all 36 specimens studied with median total GR levels in blood, bone marrow, and lymph nodes being 3018 (n = 17), 3524 (n = 12) and 3102 (n = 7) sites/cell respectively. GR levels did not correlate with in-vitro sensitivity, as measured by inhibition by dexamethasone of radiolabelled leucine, uridine, and thymidine uptake, nor with the clinical or laboratory features examined. Nine patients were treated with dexamethasone as a single agent; blood GR levels were not predictive of antitumor response. We conclude that in patients with CLL, malignant cells from all involved tissues have measurable numbers of GRs, but these are not correlated with in-vitro sensitivity to glucocorticoid. Blood GR levels do not correlate with antitumor response; further studies are required to see if bone marrow or lymph node receptor levels correlate with antitumor response to steroids.

Enzyme-linked antibody was used to treat unfixed herpesvirus-infected human fetal lung cell cultures in a mode which permitted the visualizing of local sites of infection. Foci containing as few as 20 herpesvirus-infected cells produced sufficient viral mass to be easily detectable by this method. 'Clouds' or 'plumes' of colored reaction product diffused into the substrate overlay, accumulated above and around each focus of infection and allowed quantitation of the number of foci in a culture. The number of minute centers of viral infection determined by the enzyme-linked antibody method corresponded almost exactly with values obtained by fluorescence microscopy. Quantitation of herpes simplex infectivity by focus assay was possible within only 17 h after culture inoculation, well before cytopathic effects were visible macroscopically. The technique was also applied to demonstrate measles and mumpsvirus plaques (infectious centers) in Vero cell cultures.

Prognostic factors for acute lymphocytic leukemia (ALL), including initial white blood count (WBC), age at diagnosis, sex, cytology, lymphocyte surface markers, and glucocorticoid receptors, have been evaluated in 12 patients presenting with ALL and the Philadelphia chromosome (Ph 1 + ALL). Ph 1 + ALL patients had a median initial WBC of 89 × 10 9 liter, a median age of 23 years and a male: female ratio of 1:1. The cytology of the blasts was L1 in 50% and L2 in 50%. The blasts in all cases studied were characteristic of non-T, non-B, common ALL, had high terminal deoxynucleotidyl transference levels, and low numbers of glucocorticoid receptors. The prognostic factors in these Ph 1 + ALL patients were compared with those of a concurrent group of adults with Ph 1 − ALL. Patients with Ph 1 + ALL more commonly had unfavorable prognostic features (high WBC, older age, male sex, L2 cytology, and low glucocorticoid receptors). They also had fewer complete remissions (58 vs. 100%, p=0.07), shorter remission durations (median 10 vs. 44+ months, p=0.001) and shorter survival times (median 11 vs. 48.5+ months, p=0.00008). However, even when patients with similar prognostic features were compared, those with the Ph 1 had fewer and shorter remissions and shorter survival times than ALL patients without the Ph 1 . These data suggest that the Ph 1 is an independent unfavorable prognostic factor in adults with ALL.

The mechanism of the lymphoproliferative effect of the macrophage product lymphocyte-activating factor [LAF(IL1] appears to be mediated by the stimulation of the release of T cell growth factor [TCGF(IL2)] by T cells. The magnitude of the resultant T cell proliferative clonal expansion is thus dependent upon the quantity of both LAF(IL1) and TCGF(IL2) induced by antigen or lectin stimulation. These observations, coupled with the ability to measure the production and actions of these hormone-like lymphokines, should allow for increased insight into the mode of action of immunoenhancing and immunosuppressive agents, as well as for new therapeutic approaches to disease states involving T lymphocytes.

A method is described for the simultaneous radioimmunoassay (RIA) for antibody to members of the human herpesvirus group. The RIA is compared with some of the conventional serologic techniques used to quantitate antibody to these viruses (Epstein-Barr virus, cytomegalovirus, herpesvirus type 1 and varicella-zoster virus). Color-coded beads, each coated with the antigens of a different herpesvirus, were simultaneously placed in a well which contained a human serum to be assayed for antibody to each of these 4 viruses. The results of this test were compared with the results obtained when the serum was assayed for antibody to the 4 viruses in 4 separate tests. We conclude that the antigen-antibody reactions do not significantly interfere with each other when a serum is assayed for antibody to the 4 viruses simultaneously. A comparison of the RIA with conventional serologic techniques shows excellent correlation in the antibody titers obtained. Features of the solid-phase RIA allow significant savings of time, reagents and space, and thus make it feasible for the small laboratory to screen large numbers of sera for antibody to a variety of antigens.

Interleukin-2 (IL-2), the first of a series of lymphocytotrophic hormones to be recognized and completely characterized, is pivotal for the generation and regulation of the immune response. A T lymphocyte product, IL-2 also stimulates T cells to undergo cell cycle progression via a finite number of interactions with its specific membrane receptors. Because T cell clonal proliferation after antigen challenge is obligatory for immune responsiveness and immune memory, the IL-2-T cell system has opened the way to a molecular understanding of phenomena that are fundamental to biology, immunology, and medicine.

A series of substituted uracil analogues of the antiviral agent 9-[[2-hydroxy-1(hydroxymethyl)ethoxy]methyl]guanine (BIOLF-62) has been synthesized and characterized. The uracil analogues show little activity against herpesviruses.

A cell line established from a patient with acute lymphoblastic leukemia was found to express IL-2 binding sites with a novel, intermediate affinity compared with the characteristic high-affinity IL-2-receptors and low-affinity IL-2 binding sites described previously. Clones were isolated from this cell line that displayed solely this new IL-2-binding protein, and were found to be unreactive with anti-Tac, the mAb that competes with IL-2 for binding. Moreover, these same cloned cells did not express mRNA detectable by hybridization with radiolabeled cDNA encoding the Tac protein. In contrast, the original cell line and similar clones expressed low levels of Tac mRNA and cell surface Tac antigen, both of which could be augmented by exposure to medium conditioned by adult T leukemia cell lines. Particularly noteworthy, induction of Tac antigen expression was paralleled by an increase in the number of high-affinity IL-2-R detectable. Since the expression of the Tac antigen protein by itself makes only for low-affinity IL-2 binding, these data prompted a reevaluation of the structural composition of high-affinity IL-2-R. Analysis of the IL-2-binding proteins expressed by leukemic cell lines lacking high-affinity receptors revealed only a single protein, larger than the Tac antigen protein (Mr = 75,000 vs. 55,000). In contrast, clones induced to express high-affinity receptors had clearly both of these IL-2-binding proteins. Moreover, when IL-2 binding to normal T cells was performed under conditions that favored the proportion of high-affinity receptors occupied, two distinct proteins identical to those already identified on the leukemic cells could be crosslinked covalently to radiolabeled IL-2. The interpretations derived from these varied, assembled data, point to two IL-2-binding proteins, both of which are required for high-affinity IL-2 binding.

The development of cytotoxic effector cells through primary allogeneic mixed tumor-lymphocyte culture (MTLC) was found to be accompanied by the production of T cell growth factor (TCGF). Addition of supplemental TCGF to MTLC resulted in the generation of significantly greater quantities of effector cells, and these effector cells displayed augmented cytotoxic activity. The TCGF-induced effect could not be duplicated by the addition of fresh medium or a mitogenic concentration of concananvalin A. Although TCGF augmented the proliferation of antigen-nonreactive cells, antigen-reactive cells appeared to be preferentially stimulated by TCGF. Finally, it was shown that depletion of TCGF from MTLC resulted in an impairment of proliferation and differentiation of cytotoxic effector cells. These findings demonstrate that soluble factors are involved in the regulation of in vitro cell-mediated immune responses in an analogous manner to similar factors that have been shown to regulate humoral immune responses. Therefore, the forces affecting TCGF production may modulate the amplitude of a T cell-mediated cytolytic response.

The previous communication presented data detailing that glucocorticoid hormones exhibited slight but significant inhibitory effects on the proliferation of cytolytic T cells maintained in long-term T cell growth factor (TCGF)-dependent cultures. However, most importantly, glucocorticoids were shown to inhibit T-lymphocyte proliferation via a profound effect on TCGF production. Addition of exogenous TCGF to glucocorticoid-treated human, mouse, or rat T cell mitogen-stimulated lymphocytes restored normal levels of proliferation. These observations led us to propose that the immunosuppressive effects of glucocorticoid hormones were mediated by controlling the production of the T cell proliferation-inducing agent, TCGF. Results of experimentation detailed in this communication provide further evidence in support of this hypothesis. We found that although glucocorticoid hormones had little effect on the cytolytic reactivity of T cells harvested from TCGF-dependent culture, treatment of mixed lymphocyte cultures (MLC) with identical glucocorticoid hormone concentrations completely abrogated in situ TCGF production and alloantigen-directed cytolysis. The inhibitory effects of glucocorticoids in MLC could be eliminated once again simply by the delayed addition of exogenous TCGF. These observations coupled with those detailed in the previous report provide evidence that, rather than effecting lysis of potentially reactive mature lymphocytes, glucocorticoids influence the development of T cell-mediated immune reactivity by inhibiting TCGF production, which in turn serves to inhibit the clonal expansion of activated T cells.

A process is described in which surface attached living cell cultures are treated, cooled, stored and revived in such a manner as to retain their viability and their adherence to culture surfaces.

Virus infected, fixed, replicate monolayer cell cultures can be pre-tested, stored, then withdrawn for use in the radioimmunoassay (RIA) as solid phase viral antigens. Ready availability of uniform, potent viral antigens permits quantitative determination of viral antibody concentrations simply, rapidly and precisely. Where cell destruction by cytolytic viruses might prevent in situ fixation of cell monolayers, a rapid and simple RIA procedure is described which utilizes imitation pearls as antigen adsorbents.

T lymphocyte mitosis results from the interaction of interleukin 2 (IL-2) with specific receptors that appear only after appropriate immune stimulation. To assess the potential role of IL-2 receptor levels in determining the rate and magnitude of T cell proliferation, the expression of IL-2 receptors by lectin-stimulated human peripheral blood T cells was examined and correlated with T cell growth. Using biosynthetically radiolabeled IL-2 and anti-Tac, a monoclonal antibody that blocks IL-2 receptor binding, IL-2 receptors were found to accumulate slowly and asynchronously among lectin-stimulated T cells and to precede the onset of DNA synthesis. Moreover, a critical threshold of IL-2 receptor density appeared to be required before the commitment to cell cycle progression, as analyzed quantitatively by tritiated thymidine incorporation and flow cytometric analysis of cellular DNA content. Once maximal IL-2 receptor expression occurred, continued proliferation was IL-2 concentration dependent as assessed using homogenous immunoaffinity-purified IL-2. Upon removal of the activating lectin, IL-2 receptor levels progressively declined, and, in parallel, the rate of proliferation diminished. The decay of IL-2 receptors could not be attributed to IL-2-mediated down-regulation. Instead, renewed IL-2 receptor expression was dependent upon the reintroduction of the initial activating signal. Repetitive exposure to lectin resulted in a more rapid reexpression of maximal IL-2 receptor levels, which was then followed by an accelerated resumption of proliferation. Thus, the extent of T cell proliferation after immune stimulation depends upon the interplay of the IL-2 concentration available and the density of IL-2 receptors expressed, both of which are ultimately determined by antigen/lectin stimulation. The awareness of the transience and the antigen/lectin dependence of IL-2 receptor expression, together with the capacity to monitor T cell cultures for IL-2 receptor levels, should facilitate the initiation and maintenance of cloned, antigen-specific T cells in long-term culture. In addition, these findings suggest that, in vivo, the rapidity of acquisition of maximum IL-2 receptor levels by activated T cells and the duration of IL-2 receptor expression may well direct the magnitude of T cell clonal expansion and resultant immune responses.

The chemical synthesis of 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine is described. This compound, known as BIOLF-62, is active against herpesviruses. This compound is a member of a novel class of nucleoside analogues which lack a rigid carbohydrate ring, but which possess all of the functional groups of naturally occurring deoxynucleosides.

Molecular biological techniques have revealed the interleukin-2 receptor to be a dimer composed of one α-subunit and one β-subunit which interact non-covalently in a cooperative manner. Site-directed mutagenesis, in conjunction with structural analysis, is beginning to clarify the relationship between structural components of the receptor and their function, and Thomas Ciardelli and Kendall Smith explain why this is bringing drug developers closer to the design of IL-2 agonists and antagonists .

Cycochemical, morphological, immunologic, and cytogenetic studies were carried out on hand-mirror cells (HMC) from a mentally retarded patient with a constitutional chromosome abnormality, 46,XX,r(21), and acute lymphoblastic leukemia. Scanning electron and differential interference contrast microscopy showed microspikes on the uropodia, but little evidence of cellular motility, despite formation and disappearance of individual uropodia in cell suspensions. The cells rosetted with sheep erythrocytes, suggesting T-cell origin. Cells derived from the bone marrow (80% HMC) showed a high degree of polyploidy (60%) and a bimodal chromosome number of 49 (49,XX,+10,-21, +3 rings) and 94 (6 no. 10, 3 no. 18, 2 no. 21 chromosomes, 3 ring chromosomes, plus 4 copies of each other chromosome).

In studies to determine the biochemical mechanisms responsible for cell proliferation, synchronized T cells were used as a model for cellular growth control. By metabolic and morphologic criteria, it was found that activation of the T-cell antigen receptor rendered the cells responsive to interleukin-2 (IL-2), but did not move them through the cell cycle. Instead, IL-2 stimulated G1 progression to S phase, or lymphocyte "blastic transformation." During IL-2-promoted G1 progression, expression of the cellular proto-oncogene c-myb was induced transiently at six to seven times basal levels, maximal levels occurring at the midpoint of G1.

The structural significance of C-terminal amphiphilic alpha-helix of human interleukin-2 has been investigated using principles of protein design. Employing disulfide-mediated semi-synthesis, several multiple residue substitution patterns were studied in order to provide rapid insight into the most appropriate features to incorporate into fully recombinant proteins. Substitutions directed toward both stabilization and destabilization of the helix resulted in proteins with modulated bioactivity. Circular dichroism verified the conformational integrity and thermal stability of the derivatives. The biologic characteristics of each derivative were evaluated in the standard murine CTLL-2 assay and compared to activities exhibited in both human T-cell bioactivity and binding assay. A strategy for the design of protein ligand agonists and antagonists without knowledge of receptor contact residues is discussed.

N-Substituted Glyceropurines have been prepared. The N-dimethylaminomethyleneated and N-acetylated glyceroguanine derivatives have significant activity against Herpesviruses.

To examine directly the hypothesis that T cell growth factor (TCGF) interacts with target cells in a fashion similar to polypeptide hormones, the binding of radiolabeled TCGF to various cell populations was investigated. The results indicate that TCGF interacts with activated T cells via a receptor through which it initiates the T cell proliferative response. Internally radiolabeled TCGF, prepared from a human T leukemia cell line and purified by gel filtration and isoelectric focusing, retained biological activity and was uniform with respect to size and charge. Binding of radiolabeled TCGF to TCGF-dependent cytolytic T cells occurred rapidly (within 15 rain at 37 degrees C) and was both saturable and largely reversible. In addition, at 37 degrees C, a receptor- and lysosome-dependent degradation of TCGF occurred. Radiolabeled TCGF binding was specific for activated, TCGF-responsive T cells. Whereas unstimulated lymphocytes of human or murine origin and lipopolysaccharide-activated B cell blasts expressed few if any detectable binding sites, lectin- or alloantigen-activated cells had easily detectable binding sites. Moreover, compared with lectin- or alloantigen-activated T cells, long-term TCGF-dependent cytolytic and helper T cell lines and TCGF-dependent neo-plastic T cell lines bound TCGF with a similar affinity (dissociation constant of 5-25 pM) and expressed a similar number of receptor sites per cell (5,000-15,000). In contrast, a number of TCGF-independent cell lines of T cell, B cell, or myeloid origin did not bind detectable quantities of radiolabeled TCGF. Binding of radiolabeled TCGF to TCGF-responsive cells was specific, in that among several growth factors and polypeptide hormones tested, only TCGF competed for binding. Finally, the relative magnitude of T cell proliferation induced by a given concentration of TCGF closely paralleled the fraction of occupied receptor sites. As the extent of T cell clonal expansion depends on TCGF and on the TCGF receptor, the dissection of the molecular events surrounding the interaction of TCGF and its receptor that these studies permit, should provide new insight into the hormonelike regulation of the immune response by this lymphokine.

A method was developed for the simultaneous transfer of large numbers of solid-phase adsorbents in radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Specially coated ferromagnetic spheres (beads) were used as the solid phase. These beads were transferred from a reaction mixture through a rinse bath to another reaction mixture by magnetic probes. The quality of results obtained with this new methodology compared favorably with that obtained when solid-phase adsorbents were handled individually. The magnetic transfer system provided a high degree of mechanization, thus permitting many more tests to be performed at one time, under almost identical conditions. Certain mechanical features of the test and the micro scale of the reactions provided substantial economy in time and consumption of valuable reagents. The sensitivities of the RIA and ELISA for detection of viral antibody were about the same. An important advantage in using magnetic devices for transfer of beads is that the immune and enzyme-substrate reactions can be started and stopped instantly in either system.

Levels of elastin production in explant culture of fetal sheep nuchal ligament and corresponding levels of translatable elastin mRNA were determined in parallel studies during a period of rapid growth of the embryo. The identity of the explant culture and cell-free products was confirmed by peptide mapping, immunoprecipitation, and the characteristic lack of histidine and methionine. Elastin production was quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioimmune precipitation. The translation products could be labeled with methionine only when NH2-terminally donated as f-Met-tRNAfMet. Explant cultures showed a large rise in elastin production from 70 days after conception to 150 days after conception. Cell-free translation of RNA demonstrated a parallel in elastin mRNA levels and in elastin mRNA per cell. It appears, therefore, that the marked emphasis the differentiating nuchal ligament places on elastin production is modulated, at least in part, by the quantities of available elastin mRNA.

The synthesis of a series of purine analogues of the acyclonucleoside compound A* (A-Star, 1) is described. Compounds in this series have been shown to have pronounced activity against herpesviruses. These compounds have been designated "the glycerosides". The glyceropurines are described in this report. Nucleotides have been constructed containing glyceroadenine (A*, compound 1). These nucleotides are resistant to degradation by phosphodiesterases. The compound A* is both a poor substrate and a poor inhibitor of adenosine deaminase.

5-Substituted cytosine analogues of 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine (BIOLF-62) have been synthesized and tested against HSV-1.

A rat X mouse T cell hybrid (PC60) proliferates in the absence of T cell growth factor (TCGF) and its cytolytic activity can be induced by culture in mixed leukocyte culture supernatants or concanavalin A-activated rat spleen cell supernatant (CS) to lyse 51Cr-labeled tumor target cells. To characterize the factor(s) responsible for this reversible induction, serum-free CS was fractionated by reverse phase high performance liquid chromatography and by phenyl-Sepharose chromatography. A cytotoxicity-inducing activity (CIA) was separated from TCGF and macrophage-activating factor/interferon-gamma. CIA was found to be a macromolecule with an apparent molecular weight of 12,000-18,000 and a pI of 5.0 and 6.2. Its activity on PC60 cells depended on the addition of TCGF. Thus TCGF may have other effects on T cells than the induction of entry into cell cycle. The number of TCGF surface receptors on PC60 cells was measured using purified 3H-TCGF. TCGF receptors were undetectable on noninduced cells but appeared during induction. The expression of TCGF receptors was not induced either by TCGF or by CIA-containing supernatants or fractions alone, only by a combination of both. These results show that TCGF plays a role in the regulation of the expression of its own receptors.

Long-term cultures of human cytotoxic T-cell lines (H-CTLL) were established. H-CTLL cells were strictly dependent on growth upon a T-cell growth factor (TCGF) produced by phytohemagglutinin-stimulated normal human peripheral blood lymphocytes. H-CTLL cells were maintained in TCGF-dependent exponential proliferative culture for over 4 mo during which time they continued to mediate stimulator antigen-specific cytotoxicity as measured by a 4-h 51Cr-release assay. H-CTLL cells recovered from cryopreserved stocks and re-established in long-term culture demonstrated similar high levels of antigen-specific cytotoxicity. H-CTLL cells were 95--100% E-rosette positive and expressed normal T and Ia-like cell surface markers. The ability to sustain differentiated antigen-specific T-effector cells in long-term culture may provide a new means for the study of both the mechanism and regulation of T-cell-mediated immunity.

We recently reported that the spleen focus-forming virus [(SFFV), a replication-defective leukemogenic virus] coded for a cell surface neoantigen which could be detected by lymphocyte-mediated cytolysis. Because the SFFV genome has recently been characterized as a recombinant between both ecotropic and xenotropic viral information, we examined whether the SFFV-specific neoantigen, as defined by in vitro cytolysis, shared determinants with antigens coded for by mink cell focus (MCF)-inducing viruses (also xenotropic/ecotropic recombinants). As detected by direct cytolysis of SFFV- and MCF-infected target cells, the SFFV-specific neoantigen was found to be partially cross-reactive with determinants on the surface of MCF virus-infected cells. The cross-reactive determinants appeared to be coded for by the envelope region of both AKR and Moloney MCF viruses, in that antisera specific for MCF gp 70 were capable of blocking SFFV-specific neoantigen-directed cytolysis. These results provide further evidence in support of the recombinant nature of the SFFV genome and its close relationship to MCF viral genetic information. However, in that the SFFV-specific neoantigen was not totally cross-reactive with MCF gp70, it remains possible that additional antigenic determinants associated with SFFV-encoded transformation may be present.

T lymphocytes are stimulated to proliferate in an autocrine/paracrine manner by the lymphokine interleukin 2 (IL-2). In seeking further insight into the mechanisms by which IL-2 induces progression of T cells through the G1 phase of the cell cycle, studies were performed with agents that increase cellular adenosine 3',5'-cyclic monophosphate (cAMP), a well-known inhibitor of lymphocyte growth. The addition of dibutyryl-cAMP, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine to an IL-2-dependent murine T-cell line evoked a dose-related suppression of S-phase transition without affecting cellular viability. Moreover, elevation of cAMP levels led to an accumulation of uniformly small cells, suggesting an arrest in early G1. Consistent with these findings, dibutyryl-cAMP inhibited the incorporation of both [3H]-uridine and [3H]thymidine by IL-2-stimulated, synchronized normal human T cells. Furthermore, maximal inhibition occurred during early G1, as indicated by experiments where the addition of dibutyryl-cAMP was delayed with respect to IL-2 stimulation. Quantitative flow cytometric analysis of RNA and DNA content of IL-2-stimulated cells affirmed that increased cAMP inhibits RNA accumulation and S-phase transition. In addition, exposure of IL-2-dependent, asynchronously proliferating normal human T cells to dibutyryl-cAMP resulted in uniform growth arrest in early G1, the point at which cycling T cells accumulate when they are deprived of IL-2. These results indicate that increased cAMP inhibits G1 progression stimulated by IL-2 and provide a rationale for the use of cAMP analogues as pharmacologic probes for the dissection of molecular events occurring during IL-2 signaling and T-cell G1 transit. They also suggest the possibility of therapeutic immunosuppression by a combination of agents that act at different stages of the T-cell cycle.

Modifications were made which increased the sensitivity of a solid-phase radioimmunoassay for serum antibody to human herpesviruses. Quantitation of antibody can be accomplished by referring to standard curve titrations.

High-affinity IL-2-R binding results from an exceptional type of cooperative interaction between two IL-2-binding proteins termed alpha and beta. When expressed together on the cell surface, these two distinct chains form a noncovalent kinetic hybrid receptor complex that exploits a rapid association rate contributed by the p55 beta chain and a slow dissociation rate characteristic for the p75 alpha chain. The p75 alpha chains signal cell growth, whereas the p55 beta chains only facilitate IL-2 binding by serving as helper binding sites, having no discernible signaling role themselves. The unique functional implications of this structural organization indicate that this cooperative bimolecular arrangement reflects a general mechanism by which the efficiency of surface receptors can be enhanced markedly.

This investigation was conducted in an attempt to determine if a reported case of disseminated intravascular coagulation (DIC) in a patient receiving polyriboinosinic-polyribocytidylic acid [poly (I) · poly(C)] could be related to an effect of poly (I) · poly (C) on platelet aggregation. It was determined that poly (I) · poly (C), while not inducing platelet aggregation by itself, produced a decrease in latency and an increase in the rate of platelet aggregation induced at 37° and 24°C by acid-soluble collagen (ASC). All activity resided in the poly (I) strand, which produced a significant effect in a concentration as low as 0.1 μg/ml of plasma (0.22 μmole phosphorus/liter). At concentrations up to 0.25 mg/ml no activity was observed with polyriboadenylic acid-polyribouridylic acid, while polyriboguanylic acid had an inhibitory effect in concentrations down to 2.5 μg/ml (0.54 μmole P/liter). Similarly, moieties of poly (I), inosine, and inosine 3’- and 5-monophosphoric acid lacked activity. Inhibition of ASC polymerization by ascorbic acid diminished the effect of poly (I) on platelet aggregation, while poly (I) alone enhanced ASC polymerization. Since poly (I) was found not to affect platelet aggregation induced by particulate collagen, it was concluded that it enhanced ASC-induced platelet aggregation by promoting collagen polymerization. Poly (I) in high concentrations (about 0.05 mg/ml) was found to inhibit platelet aggregation induced by adenosine diphosphate, epinephrine, and ristocetin. The inhibitory effect, however, did not appear incompatible with the appearance of a thrombotic disorder since plasma concentrations in vivo probably fall rapidly below the effective inhibitory concentration. The reported presence of a soluble form of collagen (procollagen) in human serum and the identification in arterial thrombi of collagen that may be derived from this source provide a possible link between poly (I) and DIC.

Solid phase radioimmunoassay (RIA) methods for measuring autoantibodies in systemic lupus erythematosus (SLE) patients' serum were developed to improve the sensitivity and quantitative precision of these determinations. Two mechanical systems were studied: (1) acetone fixed cell monolayers in glass tubes and (2) antigen coated plastic beads. Both systems were sensitive and reproducible, giving serum dilution end-points between two and four orders of magnitude (100-10,000 times) greater than those obtained by fluorescence microscopy. The most sensitive, versatile system involves the coating of plastic beads with nuclear antigen(s), incubation overnight with sera and labelling with 125I conjugated antihuman globulin. Linear binding of this radioactive tag is obtained over a wide range of SLE serum dilutions and the slopes of the serum dilution titration curves are almost identical for all SLE patients' sera we have tested. Therefore, a standard titration curve can be constructed from the results with a positive serum, and end-point dilutions of unknown sera estimated from results obtained with a single serum dilution. Alternatively, binding ratios of unknown sera can be usefully compared at fixed dilutions with standard positive and negative sera. For example, high binding ratios (greater than 3.0) were obtained with 19/20 SLE sera and 0/20 control sera. Antigens used in these systems include crude, whole-cell lysates and lysates from purified nuclei. These RIA methods appear to provide certain advantages over existing autoantibody assay methods because they are relatively simple, sensitive, reproducible and potentially capable of measuring a variety of autoantibody specificities.

Murine spleen cells activated by concanavalin A (Con A) in culture produce a class of lymphokine molecules which possess biological activity in a number of lymphocyte response assays. Lymphokines with a mol wt of 30,000, as estimated from gel filtration studies, can be resolved into two components which differ by charge, with isoelectric point (pI) values of 4.3 and 4.9, respectively. Both components stimulate (a) the growth of established T-cell lines in culture, (b) the proliferation of thymocytes in the presence of Con A under culture conditions where Con A alone is nonmitogenic, (c) the induction of antibody responses to heterologous erythrocyte antigens in athymic (nude) spleen cultures, (d) the generation of cytotoxic T lymphocytes (CTL) in thymocyte cultures, and (e) the generation of CTL in nude spleen cultures. In each of these culture systems we suggest that the assays are detecting a single class of lymphokine which acts directly on activated T cells. Nonactivated T cells must be stimulated by either antigen or mitogen before becoming responsive to lymphokine, but do not require antigen or mitogen for continued growth with lymphokine. The two molecular species, separable by isoelectric focusing are referred to as the T-cell growth factor (TCGF). A lymphokine, similar in size (30,000 daltons) to TCGF but heterogeneous in charge (pI 3.0--4.0), stimulates immune responses to erythrocyte antigens in T-cell-depleted spleen cultures but has no stimulatory activity in the other lymphocyte assay systems described. The data have been interpreted as showing the two molecular forms of murine TCGF (pI 4.3 and 4.9) are responsible for many of the lymphokine activities described elsewhere as thymocyte mitogenic factor, nonspecific T-cell-replacing factor and killer helper factor or costimulator. The other lymphokine, separable from TCGF by charge, appears to have true T-cell-replacing activity.

We describe a series of investigations on mechanisms of antiinflammatory and immunosuppressive actions of glucocorticoids. Glucocorticoid receptors and primary mechanisms of action have been studied with isolated rat thymus cells. When added to these cells glucocorticoids immediately form cytoplasmic hormone-receptor complexes which after activation bind to the nuclei where they apparently induce mRNA for specific proteins that rapidly inhibit glucose transport and acetate incorporation into lipids. Protein and RNA metabolism are inhibited more slowly and eventually the cells die. With normal peripheral human lymphocytes, similar but slower effects are produced. A dramatic increase in receptor sites per cell is seen in lymphocytes stimulated with concanavalin A or antigen. This increase is probably associated with preparation for mitosis. We do not find, contrary to widespread belief, that these stimulated cells are insensitive to glucocorticoids. Such insensitivity has been invoked to explain the insensitivity to glucocorticoid suppression of secondary compared to primary immune responses. An alternative explanation emerges from our experiments with T-cell growth factor. T-cell growth factor, produced by mitogen or antigen stimulated spleen cells, is necessary for the proliferation of T-lymphocytes in culture and may be responsible for clonal expansion of antigen-responsive T-cells. Treatment of Con A-stimulated rat spleen cells or human peripheral mononuclear cells with 100 nM dexamethasone inhibits the production of the growth factor by 95%. This effect is specific for glucocorticoids. Addition of T-cell growth factor completely reverses the inhibition by glucocorticoid of mitogen-induced blastogenesis. We propose that the inhibitory actions of glucocorticoids on antigen- or mitogen-induced T-cell blastogenesis are related to inhibition of production of T-cell growth factor and that the reason the primary immune response is more sensitive to glucocorticoids is that by inhibiting production of T-cell growth factor, glucocorticoids block the clonal expansion necessary to amplify the primary response. We have also studied effects of glucocorticoids on Fc receptors, which play important roles in phagocytosis and other aspects of immune responses. Treatment of the human progranulocytic cell line HL-60 with dexamethasone for 72 hours reduces by 35-50% the Fc receptors per cell with no effect on cell viability or proliferation and slight increase in leucine incorporation. The effect is specific for glucocorticoids. These findings indicate that an important component in glucocorticoid-induced immunosuppression may be a reduction in Fc receptors, and with the results on T-cell growth factor, suggest that glucocorticoids regulate immune processes via effects on lymphokines and other immunologically important proteins.

To determine the effects of glucocorticoid administration on the number of measured lymphocyte glucocorticoid receptor sites and the duration of such effects, seven normal volunteers were studied. Glucocorticoid receptor levels of the lymphocytes circulating in the blood of each volunteer were determined. Glucocorticoid was then administered in a regimen of a total of four doses of dexamethasone 4 mg p.o. every 6 hr. Determinations of the number of receptors were performed at 6 hr and at various subsequent times after the end of dexamethasone administration. When compared to baseline receptor numbers, six volunteers showed a decrease in receptor number after glucocorticoid administration (median maximum decrease 2,046 sites/cell). The fall in receptor number occurred rapidly, reaching a nadir within 30 hr from the end of glucocorticoid administration. The return of receptor number to baseline was more gradual, requiring from 3 to as long as 17 days in one subject. Our results suggest that in order to accurately interpret glucocorticoid receptor numbers in human lymphoid cells, glucocorticoid should not have been administered for 3 wk prior to determinations of receptor levels.

Treatment of human sera with protein A reduced the amounts of immunoglobulin G (IgG), IgA, and IgM detected by radial immunodiffusion. This treatment also decreased the amount of herpes-specific IgG and IgM detected by radioimmunoassay, whereas it increased and even unmasked the amount of herpes-specific IgA detected. Comparison of protein A-treated sera with untreated sera indicated that herpes simplex virus type 1 IgG was responsible for more than 92 to 99% of the serum neutralizing activity.

SummaryEvidence is presented that measlesvirus antibody and herpesvirus antibody are sometimes present in human aortic tissues in a state not readily dissociable by extensive washing with buffered physiologic saline. This antibody is readily released from the tissues by high molarity potassium iodide solution, a treatment known to dissociate antigen-antibody complexes. We postulate: (1) that viral antigen-antibody complexes are sometimes present in aortic tissues; and (2) that a significant cause of vascular tissue injury in humans may be viral infection of the blood vessels themselves and/or deposit of viral antigen-antibody complexes in the vascular tissues.

The cell surface density of high-affinity membrane receptors for the T-lymphocytotrophic hormone interleukin 2 (IL-2) determines the rate of T-cell-cycle progression. Since 10-fold greater numbers of IL-2 receptor molecules were found by using a radiolabeled monoclonal antibody reactive with IL-2 receptors (anti-Tac) compared with binding of IL-2, the functional relationship of the binding sites recognized by both of these ligands was assessed. In the presence of cycloheximide, IL-2 binding sites declined with a half-time (t1/2) of 2.6 hr, whereas the decay of anti-Tac binding sites was much slower (t 1/2 = 6.4 hr). Moreover, after limited membrane proteolysis, the half-time for the reappearance of IL-2 binding sites was remarkably similar to its decay (t 1/2 = 2.2 hr), while Tac antigen reappearance was markedly retarded, returning to only 20% of original levels within 5 hr after proteolysis. Addition of homogeneous immunoaffinity-purified IL-2 to cell populations that expressed equivalent IL-2 and anti-Tac binding sites resulted in a time- and temperature-dependent 8- to 10-fold enhancement of Tac epitope expression and, simultaneously, a 20-30% diminishment of detectable high-affinity IL-2 binding sites. As the magnitude of the IL-2-dependent proliferative response correlated with the density of high-affinity IL-2 binding sites, rather than Tac antigen levels, quantitation of Tac epitope density does not provide a reliable indication of IL-2-responsiveness among activated T-cell populations. Instead, IL-2-receptor interactions actually promote the loss of IL-2 responsiveness by diminishing the density of high-affinity binding sites at the time that Tac antigen levels are increased.

Monospecific cloned cytolytic T-lymphocyte lines have been created utilizing T-cell growth factor. The clones were found to retain their cytolytic specificity after prolonged culture and monospecific function was demonstrated by subcloning procedures. Thus, detailed studies of the phenotypic and functional characteristics of monospecific, homogeneous, cytolytic T lymphocytes will now be possible.

GLUCOCORTICOIDS exert widespread suppressive effects on lymphocytes of most species1,2. Sensitivity to the hormones is thought to vary with the state of lymphocyte maturation and differentiation. Immature thymic lymphocytes, for example, are more sensitive than mature thymic-dependent lymphocytes1,3. Furthermore, in vitro studies have suggested that glucocorticoid sensitivity varies with the state of mitogen or antigen-induced activation. Nowell4 observed that prednisolone-21-phosphate reduced the number of mitoses induced in human peripheral blood lymphocytes by phytohaemagglutinin (PHA), and noted that this inhibition required the presence of glucocorticoids during the first 24 h of exposure to PHA. When glucocorticoids were added 48 h after PHA, a time when mitoses had not yet appeared, they had no effect on the number of mitoses measured 2 d later. Tormey et al.5 extended these results to show that prednisolone-21-phosphate inhibited incorporation of radiolabelled thymidine and cytidine into PHA-stimulated lymphocytes, and found that delayed addition of steroid resulted in diminished inhibition.

Neoplastic tissues from 28 adults with malignant lymphoma were examined for glucocorticoid receptors and in-vitro sensitivity to glucocorticoids. The patients were then treated with dexamethasone for 5-14 days. 13 patients achieved at least a partial remission, and 15 had no significant tumour response. Lymphoma cells from patients who responded had more glucocorticoid-receptor sites per cell and greater in-vitro sensitivity as measured by glucocorticoid inhibition of incorporation of leucine and uridine than did tumour cells from non-responders. Study of tumour glucocorticoid receptors and glucocorticoid sensitivity in vitro may allow selection of those patients with lymphoma who should receive glucocorticoids as part of combination chemotherapy.

Using a cloned cDNA copy of T-cell growth factor (TCGF) mRNA from the Jurkat leukemic T-cell line, we have isolated three overlapping TCGF genomic clones from a human DNA library. The entire TCGF gene is contained within two adjacent EcoRI fragments spanning about 8 kilobases. The complete nucleic acid sequence was determined. The gene is divided into four exons. The 5' untranslated region and the first 49 amino acids of the protein, 20 of which constitute a signal polypeptide and are not present in the secreted protein, are encoded by the first exon. Exons 2 and 3, separated from each other by a long intervening sequence, contain coding information for the next 20 and 48 amino acids, respectively. The remaining 36 amino acids and the 3' untranslated region are contained in the fourth exon. A promoter sequence T-A-T-A-A-A is present 77 base pairs (bp) upstream from the translation initiation site, and a CAT homology region occurs 104 bp upstream from the initiation site. A putative site for initiation of mRNA transcription was identified 53 bp 5' of the translation initiation codon. The organization of the gene was shown by Southern blot analysis to be identical in normal peripheral blood lymphocytes and in a variety of malignant lymphoid cell types. Restriction analysis of these cellular DNAs produced results exactly as predicted by the map for the cloned genomic TCGF, indicating that there is only a single copy of the human TCGF gene.

A solid-phase radioimmunoassay technique was used to quantitate antigen-antibody reactions between various human cell lines and lung cancer patients' sera. Four human fetal lung cell lines and four human tumor cell lines were more or less reactive as antigens. Failure to obtain exact correspondence between reactions with these cell lines indicates that more than one antigen may be required for detecting specific antibodies to the various lung tumor types. These results suggest that serum antibody detection might be a feasible approach to the immunodiagnosis of lung cancer at stages when the tumor masses are relatively small.

To design a sensitive and reproducible immunoassay for interleukin 2 (IL–2), two IL–2 reactive monoclonal antibodies (DMS–1 and DMS–5) were characterized extensively. The unique IL–2 binding characteristics of DMS 1 and DMS 5 were exploited to construct an IL–2 immunoassay that is sensitive to 10 pM murine and human IL–2 (150 pg/ml), amenable to both radioisotopic and enzymatic detection, and readily available to other laboratories.