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A comprehensive immunologic and serologic analysis was performed on 31 untreated patients with Hodgkin's disease. Immune evaluations stressed T-cell functional activity and included traditional parameters (PHA responsiveness and delayed hypersensitivity skin reactivity), as well as newer functional assays (T-cell colony formation, chemotaxis, spontaneous and antibody-dependent cytotoxicity, and concanavalin A-induced suppressor cell activity (CISA). Serum factors included ferritin, prostaglandins, zinc, copper, immune complexes, and thymic hormone activity. Every patient exhibited at least one T-cell or serum abnormality. The greatest percentage of patients exhibited T-cell defects in chemotaxis (85%), colony formation (81%). and PHA reactivity (64%). Immune defects were more common with advanced disease but were not related to absolute T-cell or monocyte count, skin test anergy, or abnormalities of T mu/T gamma cell proportions. Linear relationships were identified among abnormalities in the three assays employing mononuclear cells (PHA, colony formation, CISA) which may have reflected the inhibitory influence of monocytes present in the mononuclear cell preparations. Low serum zinc correlated with marked impairment of T-cell chemotaxis. Elevated prostaglandins were associated with high PHA reactivity and with depressed colony formation. Our results indicate that many complex factors, including intrinsic T-cell defects, contribute to the impaired immunity associated with Hodgkin's disease.

Thymic function was evaluated by quantitation of circulating thymic factor in patients with several forms of severe infantile immunodeficiency diseases. Direct quantitation of thymic factor in serum of patients with severe combined immunodeficiency revealed heterogeneity of this syndrome by this parameter, as was also shown by study of susceptibility of the marrow cells to differentiation in vitro. Thymic factor was not detectable in one patient with severe combined immunodeficiency, but was present in normal or near-normal concentrations in three others. Circulating levels of this hormonal activity were also not detectable in a patient with DiGeorge athymic syndrome. Following marrow or fetal liver transplantation, which corrected the severe combined immunodeficiency thymic factor levels either increased slightly or did not change appreciably. Fetal thymic transplantation, which together with fetal liver transplantation corrected the immunodeficiency in one patient with severe combined immunodeficiency, was associated with increase of thymic factor to normal levels. Fetal thymus transplantation alone, which was employed to correct the immunodeficiency of DiGeorge athymic syndrome, caused an increase in thymic factor activity to normal or near normal levels in this patient.

In the rosette inhibition assay, thymopoietin was active and its activity was enhanced in the presence of high concentrations of ubiquitin. Facteur thymique serique (FTS) was active in the same assay, but its activity was completely inhibited by high concentrations of ubiquitin. Mixtures of thymopoietin and FTS showed activity both in the presence or absence of ubiquitin depending on the concentration of thymopoietin or FTS in the mixtures. There seemed to be an important biologic interaction of thymopoietin and FTS when presented as a mixture, suggesting that thymopoietin and FTS might interact in vitro and possibly in vivo .

Thymosin alpha 1, an acidic 28-residue peptide, enhances immune function. We have described a radioimmunoassay for this thymic factor based on a rabbit antiserum raised against a thymosin alpha 1-(15-28) conjugate (Incefy et al., J. Immun. Meth. 1986, in press). The detailed antigenic specificity of this antiserum was determined by measuring the ability of synthetic segments and analogues of thymosin alpha 1 and related peptides to compete with radioiodinated Ac-Tyr-thymosin alpha 1-(15-28) in this radioimmunoassay. The antiserum bound segments Ac-(1-28), (15-28), (20-28) and (21-28) with nearly equal efficiency but failed to bind segments Ac-(1-10), (11-20), (19-24) and (22-28). Thus, the major immunoreactive site seen by the antiserum is the COOH-terminal segment (21-28) (Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH). Immunoreactivity of (21-28) was nearly abolished when the carboxylate groups of Glu-21, Glu-27 and Asn-28 were omitted separately. The antiserum bound to prothymosin alpha and thymosin alpha 11, which lack the alpha-carboxylate group of Asn-28, with 0.9 and 0.2%, respectively, of the efficiency of thymosin alpha 1. But it bound nonspecifically to parathymosin alpha, which contains the internal segment . . . -Glu-Val-Val-Glu-Glu-Glu-Glu-Asn- . . . . Residues Glu-21, Glu-27 and Asn-28 of thymosin alpha 1 may be important features of the antigenic site through their ability to induce helical structure, through the ability of their negatively charged carboxylate groups to bind to specific sites on the antibody or both.

Ability to form autologous rosettes (A-rosettes) is characteristic of a certain population of immature T cells, present in the thymus of various species including man. In mice, few A-rosettes are normally found in peripheral blood and spleen but their number increases markedly in spleen following thymectomy. In our studies, A-rosette formation could be demonstrated to be significantly enhanced in the spleen of C57B1/Ks mice after the animals had been maintained 3, 6, and 8 weeks on a zinc-deficient diet (Zn − ) and to increase progressively with duration of feeding the Zn − diet. These changes were quickly reversed by feeding a normal diet containing zinc but could not be eliminated by complete adrenalectomy, a finding that ruled out their dependence on pituitary-adrenal as is function attributable to stress. Pair-fed controls and mice fed a zinc-supplemented diet ad libitum showed few A-rosettes, as expected.

Thymic secretory function was assessed by determining levels of circulating thymulin-like activity in plasma of 21 pediatric patients infected with the HTLV-III/LAV retrovirus. All the patients had serum antibodies against p41 antigens of HTLV-III on Western blot analyses. In accordance with the latest definition established by the Centers for Disease Control, 14 patients had the acquired immunodeficiency syndrome (AIDS) and the remaining 7 were classified as having AIDS-related complex. Their ages ranged from 1 to 7 years, with 10 being less than 1 year of age. Circulating thymulin activity, normally highest in healthy children under 15 years of age, was undetectable in 11 patients and below normal range for age in the remaining. OKT4/OKT8 ratios of T-cell subsets in peripheral blood were below normal in the majority of patients. Our findings suggest that thymic epithelial injury may be an early event in HTLV-III/LAV-related disease and may precede the development of clinical and/or immunologic aberrations.

Levels of Facteur Thymique Serique (FTS) were measured by rosette inhibition assay in the sera of patients with mycosis fungoides (MF) and Sezary syndrome (SS). Increased levels of FTS were detected in 15 of 23 patients with MF. Two patients with SS showed normal values. The biological activity quantitated in the serum was shown by specific immunoabsorption using anti-FTS antibodies to be due to FTS and not to allogeneic factors (AF).

Summary Markers for T lymphocytes and in vitro differentiation of PBL and marrow cells under the influence of human thymic extract were studied in a patient with ataxia telangiectasia. Certain populations of cells from blood and marrow could be differentiated in normal controls and in our patient by human thymic extract to develop characteristic markers for T lymphocytes.

Serum thymic factor (facteur thymique serique, FTS) induces in vitro differentiation of T-cell precursors into more mature cells with T-cell characteristics. As isolated from porcine serum, FTS is the nonapeptide GIp-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn. We have described two radioimmunoassays that detect FTS but not other thymic hormones [Ohga et al., (1982) J. Immun. Methods, in press]. One assay is based on a monoclonal antibody from a hybridoma derived by fusion of mouse myeloma cells and spleen cells from a mouse immunized with an FTS-mouse IgG conjugate. The second assay is based on the antiserum from a rabbit immunized with FTS bound to F(ab')2 fragments of rabbit IgG. The detailed antigenic specificity of these anti-FTS antibodies was determined by measuring the ability of FTS and 12 synthetic FTS peptide analogues to compete with a radioiodinated FTS analogue in these radioimmunoassays. The mouse monoclonal antibody and the rabbit antiserum showed similar structural requirements for binding of the FTS peptides. Since FTS had been attached to the carrier proteins through the ϵ-amino group of Lys-3, both antibodies were relatively insensitive to omission of 1 or 2 N-terminal residues, replacement of Glp-1 with either Ala or Tyr-Ala, or substitution of Lys-3 with Ala. In contrast, binding of FTS to the antibodies was substantially decreased by omission of 3 or 4 N-terminal residues, omission of 2 or 3 C-terminal residues, or replacement of Gly-7 or Asn-9 with Ala. Relative to the mouse monoclonal antibody, the rabbit antiserum was more sensitive to the omission of 4 N-terminal residues and much more sensitive to replacement of Gln-5, Gly-6, or Asn-9 by Ala. Both antibodies were relatively specific for molecules ending in -Xxx-Xxx-Gly-Gly-Ser-Asn-OH, which corresponds to the biologically active region of FTS.

The effect of thymus extract on the development of cell surface antigenicity in two patients with primary immunodeficiency was studied and compared. A stem cell fraction from bone marrow preparations of a patient with severe combined immunodeficiency failed to develop cells with antigenic markers of T-lymphocytes while that of a patient with common variable form of immunodeficiency who had both severe functional deficits of T-lymphocytes and agammaglobulinemia developed cells with such markers after treatment with thymus extract. These findings indicate that the two forms of severe combined immunodeficiency disease studied have different bases which may be reflected in differences of response to differentiative influence of thymus extract.

A new radioimmunoassay (RIA) is described for the quantitation of thymosin alpha-1 (alpha-1). The assay employs an antiserum specific for the COOH-terminal half segment 15-28 of alpha-1, synthetic alpha-1-(15-28) as the hormone standard, and a radioiodinated N alpha-acetyltryrosyl-alpha-1-(15-28) as the tracer. Since alpha-1-(1-28) lacks a phenolic ring for direct radioiodination, the N alpha-acetyltyrosyl-alpha-1-(15-28) was synthesized by the solid-phase method. The peptide bears a Tyr in place of Lys in position 14 of the natural peptide. It showed full alpha-1-(15-28) immunoreactivity and its radioiodinated derivative served as tracer in the RIA. An anti-alpha-1-(15-28) antiserum was raised in a rabbit and was shown to recognize alpha-1-(15-28) or its tyrosyl analogue, and the peptide, alpha-1-(1-28). But it did not recognize other thymic hormones or the biologically active segment 32-36 of thymopoietin, or structurally unrelated peptides. It could also detect natural alpha-1 cross-reacting material in the cytoplasm of cultured human thymic epithelial cells as measured by indirect immunofluorescence. In the RIA, as little as 9 pg of alpha-1-(15-28) equivalents in a 50 microliter sample could be detected. In addition, alpha-1-(1-28)-like immunoreactivity was quantitated in 6 human thymus homogenates and ranged from 0.5 to 4.5 ng/mg of protein.

Recently it has been found that children with severe combined immunodeficiency (SCID), who are thought to lack lymphoid stem cells capable of developing into either the B- or T-lymphocyte populations, do not possess cells in their marrow which can be induced by partially purified thymic extracts to differentiate into T-lymphocytes in vitro . The initial observations were made with marrow cells from three untreated infants with SCID under the same conditions which revealed clear evidence of induced differentiation in normal individuals. Marrow cells and peripheral blood lymphocytes from two of these children were studied again after bone marrow transplantation (BMT). Clear indication of precursor cells was obtained in marrow and blood after successful transplantation. The newly acquired precursors could be differentiated in vitro into T-lymphocytes under the influence of thymic extracts. T-cell characteristics were established both with a specific anti-human T-cell serum (ATCS) and by the E-rosette technique using sheep red blood cells (SRBC). This approach reveals a new means of dissecting the immunodeficiency diseases before BMT and a method for assessing cellular reconstitution after marrow transplantation.

Publisher Summary Human hemopoietic cells can be characterized by the presence of an array of unique and shared receptors on their surface. These surface receptors can be used as differentiation markers because they have been shown to be present on cells only at specific stages of development. Thymocytes and peripheral T lymphocytes have receptors for sheep erythrocytes and thus form E rosettes. B lymphocytes, monocytes, and mature granulocytes have complement receptors and form EAC rosettes. B lymphocytes alone have receptors for mouse erythrocytes and form M rosettes. The expression of all of these markers has been evaluated on all cell fractions after incubation with or without inducers. This chapter reviews some experiments to discuss the induction of surface receptors on granulocyte, T, and B-cell precursors in human bone marrow by thymic and non-thymic agents. It provides an overview of the methods used in the experiments and the results of the experiments. These experiments suggest that the cell separation procedures provide sufficiently homogeneous cell populations to allow identification of the cells responsive to the polypeptide hormones and cyclic nucleotide studied. These agents induce the expression of the receptor for sheep erythrocytes by a direct action on lymphocytes, and the complement receptor by a direct action on immature granulocytes. Some induction of a B-cell marker was observed in the experiments, but it is not clear whether this is due to a direct effect of thymopoietin on B cells or an indirect effect via thymopoietin influence on T cells. The chapter further presents several examples of the hormonal induction of hematopoietic differentiation.

Zinc deficient humans and animals have depressed thymic mass and increased susceptibility to infection. In the present studies, we investigated the relationship between cellular immunity, thymic hormones, and serum zinc levels in 19 patients with common varied immunodeficiency. Five (26%) had serum zinc levels 2 SD below normal and 11 (58%) had abnormally low lymphocyte proliferation to at least one mitogen. A significant statistical correlation between zinc levels and lymphocyte proliferation to phytohemagglutinin and concanavalin A was identified. Forty-two percent had abnormally low levels of facteur thymique serique and 74% had low levels of thymopoietin, although no statistical relationship between the levels of these hormones, zinc levels, or lymphocyte proliferation could be identified. Three patients with the most profound zinc deficiency had substantial increases in thymic hormones after zinc repletion, and two had complete resolution of intractable diarrhea. A therapeutic potential of zinc for certain patients with hypogammaglobulinemia is suggested.

Four radioimmunoassays (RIA) are described for the quantitation of serum thymic factor (facteur thymique serique, FTS), a thymic peptide hormone. Each assay employs an antibody specific for FTS, synthetic FTS (Glp-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn) as the hormone standard, and a radioiodinated FTS analogue as the tracer. Since FTS lacks a tyrosine residue, 2 FTS analogues were synthesized by the solid-phase method with tyrosyl-alanyl or 3-(2,6-dichlorobenzyl)tyrosyl-alanyl in place of the amino-terminal pyroglutamyl residue (Glp). They showed full FTS immunoreactivity and their radioiodinated derivatives served as FTS tracers. Two assays used the antiserum from a rabbit immunized with an FTS-protein conjugate. Two other assays used a monoclonal antibody against FTS produced by a hybridoma derived from mouse myeloma cells and splenocytes from a BALB/c mouse immunized with an FTS-mouse IgG conjugate (Ohga et al., 1982). All 4 RIAs were specific for FTS. The more sensitive rabbit antiserum can detect as little as 1 pg of FTS in a 50 microliters sample, which may allow quantitation of the FTS circulating in human peripheral blood.

The effect of zinc deficiency (Zn − ) on the circulating thymic hormone (FTS) levels in A/J mice was studied. After 3 weeks of feeding the mice a Zn − diet, FTS levels were markedly reduced and after 17 weeks, FTS was undetectable. By contrast, the zinc-supplemented (Zn + ) group seemed to maintain FTS levels better than the normal diet group with aging. On the other hand, spleen spontaneous rosette-forming cells (sRFC) were studied for their azathioprine (AZ) sensitivity in A/J mice on different diets. The Zn − mice had fewer sRFC than did the normally fed or Zn + mice. The role of zinc in controlling levels of FTS and thus thymic function is discussed.

Thymosin α1 (molecular weight 3108) is one of the many active polypeptides isolated from thymosin fraction 5 (TF5) (Low and Goldstein, 1979; Goldstein et al., 1982). α1 was initially purified from extracts of calf thymus glands. Biologically active α1 has now been successfully synthesized by classical solution (Birr and Stollenwerk, 1979; Wang et al., 1978), solid-phase (Wang et al., 1980; Folkers, et al., 1980) and recombinant DNA procedures (Wetzel et al., 1980).

Transplantation of two fetal thymuses failed to reconstitute the immune function in a patient with the DiGeorge syndrome. Serum thymic hormones (facteur thymique serique and thymopoietin), which had been nondetectable, became normal after thymus transplantation. Studies done on the patient's fractionated bone marrow cells in vitro appeared to reflect defective capacity for T-lymphocyte differentiation. In spite of the efforts at treatment, the child died at 2 years of age, apparently due to an associated cardiovascular abnormality. At autopsy, no thymic tissue could be found. Failure of immunologic reconstitution after fetal thymus transplantation in this patient with the DiGeorge syndrome may have resulted from deficiencies of precursor cells.