Studies on some physicochemical properties of a thymus humoral factor conferring immunocompetence on lymphoid cells.

By means of an assay of graft-versus-host activity some properties of the thymic humoral factor which confers immunocompetence upon lymphoid cells in vitro have been studied. Allogeneic and xenogeneic thymic preparations were found to activate lymphoid cells from neonatally thymectomized mice, enabling initiation of a graft-versus-host response. Thus, this thymus factor is apparently neither strain nor species specific. The active principle of calf thymus extracts was found to be in the supernatant after prolonged ultracentrifugation. When exhaustive dialysis and ultrafiltration through Diaflo membranes were performed, the active thymus agent was found to pass through both the dialysis sac and Diaflo UM-2 membranes. The molecule which confers immunocompetence upon lymphoid cells thus seems to be of molecular weight of an order of magnitude of 1000 or less. Dialyzed thymus preparations injected into neonatally thymectomized mice also restored the capacity of spleen cells of these mice to induce graft-versus-host activity. When injected into intact mice, thymus extract also increased the proportion of competent cells in the spleens of these animals, probably by activation of target cells originating outside the spleen.

Hormone-like activity of a thymus humoral factor on the induction of immune competence in lymphoid cells.

Experiments reported here were performed to understand the mechanism by which THF increases the immunocompetence of spleen cells from NTx mice. Dibutyryl cAMP or substances which increase intracellular levels of cAMP in lymphocytes such as Poly(A:U), theophylline, or PGE(2) were shown to mimic the effect of THF and confer reactivity in an in vitro GvH response to spleen cells from NTx mice. Flufenamic acid, an antagonist to PGE(2), was shown to inhibit the induction of competence by this substance. It was found that THF induces competence by activating membranal adenyl cyclase which leads to a rise in intracellular cAMP in thymus-derived cells only. These biochemical changes occur before antigenic stimulation and are unrelated to antigenic challenge. These findings indicate that THF exerts its effect via cAMP and are in agreement with the concepts which permit to classify THF as a thymus hormone.

Contribution of a thymic humoral factor to the development of an immunologically competent population from cells of mouse bone marrow.

The hypothesis that cells located in mouse bone marrow can acquire immunological competence by a process that involves interaction with a noncellular component of the thymus was tested using an in vitro assay of graft-versus-host reactivity as a criterion of cell competence. When suspensions of C57BL bone marrow cells were incubated in thymus extract and injected into mice incapable of inducing a response in the graft-versus-host assay as a result of neonatal thymectomy, or adult thymectomy plus irradiation, or because of genetic similarity with the (C3H x C57BL)F(1) tissue used for challenge in the assay, competent cells were recovered from the spleens of the injected mice. The reactive cells were shown to be of bone marrow origin since immune reactivity was related to the genetic makeup of the bone marrow cells rather than that of the intermediate recipients. A thymic factor was involved in the process leading to immune reactivity by these cells, as bone marrow cells incubated in xenogeneic or syngeneic thymic extracts induced a graft-versus-host response after passage through nonresponsive mice, whereas incubation of bone marrow cells in xenogeneic lymph node or spleen extracts or in culture medium only did not lead to subsequent reactivity. Participation of peripheral lymphoid tissue seemed essential in this process since bone marrow cells tested directly after exposure to thymic extract failed to induce a graft-versus-host response. C57BL bone marrow cells exposed to thymus extract and cultured together with fragments of (C3H x C57BL)F(1) spleen tissue in vitro were competent to induce a graft-versus-host response; thus, these components would seem to be sufficient as well as necessary for the immunodifferentiation process leading to graft-versus-host activity. It is concluded that one step in the process by which bone marrow cells acquire competence vis-a-vis the graft-versus-host response depends upon a thymic agent that is noncellular and extractable, and that another stage in this process is under the influence of components found within the peripheral lymphoid tissue environment. It is suggested that differentiation of precursor cells to competence could occur by progressive development of the cells in separate compartments of the lymphoid system.

Studies on characterization of the lymphoid target cell for activity of a thymus humoral factor.

The immune response to SRBC was measured in the spleens of adult thymectomized, total body irradiated mice injected with various combinations of thymus and bone marrow cells together with thymic humoral factor (THF). It was found that the number of plaque-forming cells was significantly increased when THF was given in vivo immediately after thymus cell administration or when thymus cells were incubated in THF before injection. On the other hand, bone marrow cells equally treated did not manifest any T cell activity, since THF-treated bone marrow cells were not able to substitute thymus cells in the system used. The results accumulated in the present experiments indicate, therefore, that the target cells for THF activity are thymus cells which acquire a higher T helper cell capacity after THF treatment.

Increased reactivity of mouse spleen cells sensitized in vitro against syngeneic tumor cells in the presence of a thymic humoral factor.

Unprimed mouse spleen cells cultured in vitro on syngeneic tumor cell monolayers have been previously shown to become specifically sensitized and to mediate cytotoxicity against the same type of tumor cells. This complete in vitro system of cell-mediated response has been presently used to test the effect of a thymic humoral factor (THF) upon the differentiation process leading to the generation of specifically committed lymphocytes. Culture media were supplemented with 2% THF during either the sensitization or effector phase, or both phases of the reaction. Whereas the addition of THF during both phases or during sensitization only resulted in a significant increase in the cytotoxicity index, THF added during the effector phase was ineffective. The behavior of unsensitized spleen cells and of spleen cells sensitized against nonrelated transplantation antigens remained unmodified by THF. After showing that the entire reaction is mediated by lymphocytes of thymic origin, THF was directly tested on T or B spleen cells. It was found that only T cells reacted to THF by an increased cytotoxic capacity, while B cells remained inactive after addition of THF. It was therefore concluded that THF activates a postthymic population of lymphoid cells, transforming them into fully competent lymphocytes.

Immunocompetence of spleen cells from neonatally thymectomized mice conferred in vitro by a syngeneic thymus extract.

Impaired immunological competence of spleen cells from neonatally thymectomized C57B1/6 young adult mice was apparent when these cells were tested in an in vitro graft-versus-host assay. Spleen cell inocula prepared from thymectomized mice did not induce enlargement of (C3H/eb x C57BI/6)F(1) newborn spleen explants, whereas the same number of cells from intact donors consistently initiated splenomegaly. Spleen enlargement was observed, however, when the explants were challenged by cells from thymectomized donors in the presence of syngeneic thymus extract, indicating that the spleen cells in suspension attained immunological competence under the influence of a non-cellular component of the thymus. Immunocompetence was also evident when the cells from thymectomized donors were first incubated with thymus extract for 1 hr and subsequently tested for reactivity. Cells from the same thymectomized donor mice exposed in parallel to extracts from syngeneic spleen or mesenteric lymph node at an equivalent protein concentration did not initiate a graft-versus-host response. These experiments demonstrate that immune reactivity in the graft-versus-host response involves activation of lymphoid cells by a humoral factor of the thymus acting directly upon these cells.

Enrichment of in vitro and in vivo immunologic activity of purified fractions of calf thymic hormone.

Thymus humoral factor (THF), a thymus hormone which participates in the processes leading to acquisition of immunocompetence of lymphoid cells has been isolated in our laboratory by a stepwise gel filtration through various Sephadex columns. THF so isolated appears to be a polypeptide of 3,000 mol wt which contains approximately 30 amino acid residues. Here we have tested the biological activity of THF fractions of successive degrees of purity upon lymphoid cells from both intact and neonatally thymectomized mice. The lymphoid cell populations were treated with the various THF fractions by in vitro incubation for a short time and by repeated injection in vivo. The treated cells evidenced increased ability to react in the graft-versus-host assay in vivo and in mixed lymphocyte cultures in vitro concomitantly with the rise of intracellular cAMP. On the other hand no activity whatsoever was shown by any of the control materials tested. These bioassays permitted isolation of fractions progressively more active than the original crude dialyzate of thymus extract tested. Thus the active peptide component of THF eluted from DEAE Sephadex A-25 column was estimated to be 2 X 10(4)-fold more active than the crude dialyzate of thymus extract which served as a starting material.

Immunopotentiating and immunotherapeutic effects of thymic hormones and factors with special emphasis on thymic humoral factor THF-gamma2.

The essential role played by the thymus in the development of the immune response was well documented in many publications. These findings prompted a long series of studies devised to define the factors produced and secreted by thymus cells, which are involved in the development and nature of immunological responsiveness. First experiments done with crude thymus extracts were followed by isolation of purified products and finally by chemical characterization and synthesis of immunologically active thymus-derived peptides. In this article we review the various thymic hormones and factors described, that is, thymosin fractions 5, the thymosins, prothymosin alpha, thymulin (FTS-Zn), thymopoietin, thymostimulin (TP-1), Thymic humoral factor (THF), and THF-gamma2. Studies demonstrating the activity of the various thymic factors in increasing the immunocompetence potential in both in vitro and in vivo conditions are discussed. The immunostimulatory potential of thymic factors was also investigated in experimental models where beneficial therapeutic effects were sought in a situation of immunological malfunction. The last part of the review is dedicated to clinical trials with thymic factors that revealed improvement in the immunocompetence potential in cases of immunodeficiencies, viral infections, and cancer and its correlation with therapeutic effectiveness. It seems that more research is required in order to better define conditions for the use of thymic factors in immunotherapy.

The role of a thymus humoral factor in the proliferation of bone marrow CFU-S from thymectomized mice.

The colony forming capacity of bone marrow cells from thymectomized mice was shown to be reduced as compared to that of bone marrow cells from normal donors. A further indication of changes in the proliferative capacity of colony forming cells (CFU-S), following thymectomy, was given by examination of the sensitivity of these cells to chlorambucil and by 3H-thymidine 'suicide' experiments; both showed that CFU-S from thymectomized mice were not cycling at the same rate as normals. It was also found that in late pregnancy of thymectomized females, there is an elevation in the number of bone marrow CFU-S and an increase in cell cycling. Such an increase could also be achieved by implantation, into thymectomized mice, of thymus lobes in closed diffusion chambers. Finally, in vitro administration of a thymus hormone (THF) reversed the suppressive effect of thymectomy on DNA synthesis in bone marrow CFU-S. Since the action of THF was restricted to bone marrow cells of thymectomized mice it is plausible that normal bone marrow contains at least two subpopulations of CFU-S, one of which is dependent upon a humoral product of the thymus.

Impaired radioprotective capacity and reduced proliferative rate of bone marrow from neonatally thymectomized mice;.

The colony forming capacity of bone marrow from neonatally thymectomized mice is reduced in comparison with that of normal animalsmin addition to this quantitative change, we observed that the bone marrow of thymectomized animals has a reduced radioprotective effect upon inoculation into lethally irradiated recipient mice. It was also found that the cellularity of spleen colonies derived from bone marrow of thymectomized animals is lower than that of intact controls. In vitro uptake of 3H-thymidine into cells of spleen colonies, and rate of DNA synthesis measured in vitro were found to be reduced in cells derived from bone marrow of thymectomized donors; The initially observed reduction in colony forming capactiy of bone marrow from neonatally thymectomized mice could be reversed by thymus reimplantation=

Potentiation of myeloid colony-formation in bone marrow of intact and neonatally thymectomized mice by the thymic hormone THF-gamma 2.

The effect of the thymic hormone THF-gamma 2 on committed stem cells of bone marrow (BM) origin was determined using the myeloid progenitor cell clonal assay. Preincubation of normal BM cells with THF-gamma 2 for 1 hour or 18 hours caused a 2- to 6-fold increase in the number of myeloid colonies in the presence of suboptimal concentrations of colony-stimulating factor (CSF). The optimal dose of THF-gamma 2 causing this enhancement was in the range of 25 to 100 ng/mL. THF-gamma 2 was not able to replace CSF as an inducer in these experiments. THF-gamma 2 neither induced IL-6 activity upon 24-hour incubation with bone marrow cells nor enhanced LPS-induced IL-6 secretion by bone marrow cells in vitro. Neonatal thymectomy (NTx) of Balb/c mice caused a decrease in myeloid progenitors, which was repaired by serial injections of THF-gamma 2. The repair of the stem cell compartment in the bone marrow correlated with an increased percentage of Thy1+ cells in the spleen of THF-gamma 2-treated NTx mice. These findings indicate that THF-gamma 2 is able to regulate committed stem cell functions in the bone marrow of immune-deprived NTx and of normal mice.

Selective accumulation of lymphocyte precursor cells mediated by stromal cells of hemopoietic origin.

Thymocytes were propagated in long-term cultures supported by stromal cells of both bone marrow and thymus origin. Interleukin 2 (IL-2) supplementation augmented the cell yield and allowed detailed phenotype analysis. Within 2-3 months of culture a cell population was selected in which the expression of Thy-1 antigen persisted, CD4 and CD8 antigens gradually declined, and Pgp-1 antigen, found on less than 5% of fresh thymocytes, was strongly increased. This cultured cell population (Thy-1.2 origin) contained no detectable spleen colony-forming units (CFU-S) but efficiently repopulated the thymus of Thy-1.1-irradiated congenic mice, indicating the precursor T-cell nature of the population. Upon removal from the stroma, the T cells exhibited poor cytotoxicity towards syngeneic tumor cells. Further propagation with IL-2 in the absence of stroma resulted in the acquisition of cytotoxic ability. Replacement of the horse serum used in the above experiments with fetal calf serum resulted in accumulation of cells expressing B220 antigen. This experimental model provides the means to maintain lymphocyte precursor cells in long-term culture and to further study their differentiation in the absence of stroma, both in vitro and in vivo.

Thymic humoral factor-gamma 2, an immunoregulatory peptide, enhances human hematopoietic progenitor cell growth.

Thymus humoral factor-gamma 2 (THF gamma 2), an octapeptide important for T-lymphocyte regulation, was assessed for its effect on the in vitro growth of human hematopoietic progenitor cells. This was achieved using a recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF)-stimulated myeloid cell colony formation (granulocyte-macrophage colony-forming cells, GM-CFC) assay as well as a recombinant erythropoietin (rEpo)-stimulated erythroid burst formation (erythroid burst-forming units, BFU-E) assay. Cells were obtained from bone marrow (BM) and peripheral blood (PB) of normal healthy donors and from patients with suppressed bone marrows. The latter group included aplastic anemia, leukemia, and lymphoma patients and patients with solid tumors who responded to intensive chemotherapy with significant pancytopenia. THF gamma 2 significantly enhanced normal BM and PB GM-CFC and PB BFU-E by 2- to 2.5-fold. This effect was totally dependent on the presence of the respective growth factors, that is, rGM-CSF or rEpo, and was specifically reversed by an anti-THF gamma 2 antiserum. Furthermore, although THF gamma 2-induced enhancement of GM-CFC colony formation was not affected by lymphocyte or monocyte depletion, the augmenting effect of the peptide on BFU-E was completely abrogated in the absence of lymphocytes. THF gamma 2-induced augmented growth of progenitor cells derived from severely suppressed marrows was minimal. However, cells from moderately neutropenic patients with leukemia in remission or with lymphoma under chemotherapy responded to the peptide similarly to cells from normal donors. These results suggest a stimulatory role for THF gamma 2 on human myeloid and erythroid hematopoietic progenitor cells. They also suggest the lymphocyte dependence of BFU-E enhancement and lymphocyte independence of GM-CFC stimulation by THF gamma 2. In the former case the thymus-derived peptide may act through the induction of certain erythroid-enhancing lymphokines.

Selective decline in differentiating capacity of immunohemopoietic stem cells with aging.

Bone marrow (BM) from young (3 months) and old (24 months) C57BL/6J mice were tested for the total number of colony forming units, which remain unchanged with age. The BM from both groups was used to reconstitute syngeneic, lethally irradiated mice that were 3 months' old. The reconstituted mice were followed for a period of 12 months for their ability to generate cell-mediated responses in mixed lymphocytic cultures and cultures containing the T mitogens--concanavalin A and phytohemagglutinin. For the first 8 months, mice given BM from young or old mice responded to a similar degree. Later, cellular immune responses of the mice reconstituted with BM from old mice declined markedly compared to those reconstituted with BM from young mice, although there was no detectable difference between the two groups in the hematopoietic compartment.

Regulatory effects of thymus humoral factor on T cell growth factor in aging mice.

The effect of thymus humoral factor (THF) on T cell growth factor (TCGF) production by T cells and the association between splenic macrophages and T cells from young (2-3 months) and old (20-24 months) mice in this respect were studied. Splenocytes were divided into three groups: stimulated with concanavalin A (Con A); preincubated with THF and then stimulated with Con A; or stimulated with Con A and thereafter incubated with THF. These cells were then examined for production of TCGF. Cells treated with Con A and THF as described above were passed on nylon wool to enrich T cell populations and added to mitogen-sensitized (Con A or lipopolysaccharide) adherent splenocytes of old and young mice in the following combinations: young adherent and young T cells; young adherent and old T cells; old adherent and young T cells; and old adherent and old T cells. The results demonstrated that: (A) cells of old mice produced less TCGF than the young; (B) preincubation of splenocytes or nylon-wool enriched T cells with THF increased the production of TCGF consistently in young mice, whereas in the old a significant increase was observed only in some cases; (C) depressed TCGF activity was observed when treatment with THF to splenocytes or nylon-wool enriched T cells from young and old mice was performed after Con A stimulation, and this was also more pronounced in the young; (D) the reduced level of TCGF in the old seemed to be related to a lesion in the T cell compartment, since adherent cells from old and young mouse spleens could support TCGF production by T cells from young mice and not from old.

Inhibition of tumor growth in syngeneic chimeric mice mediated by a depletion of suppressor T cells.

Syngeneic chimeric (lethally irradiated and reconstituted with syngeneic bone marrow cells) mice manifested an increased resistance to the development of Lewis lung carcinoma. In addition, these mice had a higher response to polyvinylpyrrolidone and a reduced reactivity to T mitogens. The present findings suggest that syngeneic chimeric mice lack suppressor T cells shown to regulate the development of Lewis lung tumor and the response to polyvinylpyrrolidone. Other components of the T cell population, such as helper cells responding to sheep red blood cells or cells involved in allograft rejection, assayed in these syngeneic chimeras were found unaffected. The fact that chimeric mice are deficient in a certain suppressor T cell population whereas other T activities are normal suggests the existence of different cell lines within the T cell population.

In vitro induction of T suppressor lymphocytes in recipients of renal allografts by THF, a thymic hormone.

Lymphocyte subpopulations were determined in 13 patients, recipients of kidney allografts, 7 of them during an acute rejection episode (ARE). Monitoring of the T lymphocyte suppressor or T helper cells was performed by aid of the theophylline sensitivity test and the local xenogeneic graft-versus-host reaction (GVHR). An absence or a striking decrease of theophylline-sensitive T suppressor cells was found in all patients during ARE. Incubation of the lymphocytes of these patients with a thymic hormone, THF, raised the number of TS lymphocytes from nil or from a very low level to normal or above. The therapeutic use of THF in selected renal allograft recipients is proposed.

Thymic humoral factor, THF-gamma 2, enhances immunotherapy of murine cytomegalovirus (MCMV) infection by both CD4+ and CD8+ immune T cells.

Infection of mice with murine cytomegalovirus (CMV) presents a model for the study of the role of the immune system in the pathogenesis of human CMV. The contribution of the different spleen cell subsets in conferring curative immunocytotherapy to fatally MCMV-infected immunosuppressed mice was assessed using adoptive immunotherapy. It was found that the efficacy of passively transferred immune spleen cells is dose dependent and that the therapeutic effect can be enhanced considerably by treating donor mice with thymic humoral factor (THF-gamma 2). Polymerase chain reaction (PCR) of the donor spleen population was negative, indicating that no MCMV-DNA was transferred with the immune cells. Analysis of the donor mice after THF-gamma 2 treatment showed increased levels of CMV-neutralizing antibodies, while enhancement of natural killer (NK) activity was transient and lasted only during the early phase of the infection. FACS analysis demonstrated that treatment with THF-gamma 2 restored the size of both cell subsets CD4+ and CD8+ that were decreased following MCMV infection. It is shown that both CD4+ and CD8+ T-cell subsets participate in controlling the development of the fatal disease in MCMV-infected immunosuppressed recipients. It is suggested that the enhancement of the immunocompetence of both populations of spleen cells from treated donors is mediated in part by the restoration of Interleukin-2 (IL-2) production by THF-gamma 2.

A mouse ear reaction for assessment of human lymphocyte immunocompetence.

A mouse ear reaction for testing cell mediated immunity of human lymphocytes using the local graft-versus-host reaction assay is described. Five million human mononuclears were locally injected into the ears of immune suppressed NZW mice. The reaction mounted was quantitated by determining the 125I-Iodo-Deoxyuridine (125I-UdR) incorporation in both ears. The ratio of 125I-UdR incorporation, of the injected to that of the non-injected ear (GVHR index), 7 days after lymphocyte injection, served as an accurate measure for the extent of the reaction. Only normal human mononuclears and purified, separated normal human T lymphocytes mounted a local graft-versus-host reaction. Whereas normal human B lymphocytes, chronic lymphatic leukemia B lymphocytes, mononuclears from patients with transitional cell carcinoma of the bladder, irradiated normal human mononuclears, mouse syngeneic mononuclears, or human erythrocytes gave no positive reaction. These experiments demonstrate that this assay can be used to quantitate an in-vivo specific graft-versus-host reaction.

Treatment of murine cytomegalovirus salivary-gland infection by combined therapy with ganciclovir and thymic humoral factor gamma 2.

An optimal therapeutic regimen against primary CMV salivary-gland infection has not yet been developed. We used a murine CMV (MCMV) model system to assess the ability of combined thymic humoral factor THF-gamma 2 immunotherapy and ganciclovir (GCV) antiviral chemotherapy to eliminate detectable viral DNA from salivary glands of infected animals. Mice in different experimental groups were inoculated intraperitoneally with MCMV, treated, and then sacrificed either 2 weeks or 3 months later. To amplify and detect MCMV DNA in infected salivary-gland tissue, we developed a sensitive polymerase chain reaction (PCR) using a glycoprotein B gene primer pair that amplifies a 356 bp segment. During the acute phase of the infection, the detection of high titers of infectious virus in the salivary glands correlated with a strong PCR amplification signal. Although active virions could not be recovered from untreated animals 3 months after viral inoculation, the PCR assay detected a latent MCMV genome. Treatment with either GCV alone or THF-gamma 2 alone had little or no effect on the presence of MCMV DNA. By contrast, combined treatment with THF-gamma 2 and GCV significantly reduced the amount of salivary-gland MCMV DNA to below the limit of PCR detection. The results presented here, and experimental data from previous MCMV research in our laboratories, imply that elimination of the virus from the salivary glands could be due in part to THF-gamma 2 restoration of the various MCMV-suppressed, cell mediated immune-responses. Combining THF-gamma 2 immunotherapy and GCV antiviral chemotherapy may be an important step toward an effective therapeutic regimen that has the potential to prevent the establishment of viral latency ensuing from primary MCMV salivary-gland infection.