Thymus-dependent Lymphocytes Research Articles

Effect of malnutrition and BCG vaccination on macrophage activation in guinea pigs

Host defense functions, particularly cell-mediated immunity, may be profoundly altered by malnutrition. Activated macrophages are required as effector cells in the final stages of a successful cell-mediated immune response against microbial pathogens. Macrophage activation was assessed by measuring the activity of superoxide dismutase (SOD) in peritoneal macrophages obtained from BCG-vaccinated and non-vaccinated guinea pigs maintained on purified diets containing 8% or 25% casein. Protein deficiency, as measured by reduced hemoglobin and serum albumin concentrations, was accompanied by significant reductions in delayed hypersensitivity to purified protein derivative (PPD) and impaired lymphocyte blastogenic responses in vitro to PPD and phytohemagglutinin. In contrast, macrophage SOD activity was inversely correlated with protein nutritional status, irrespective of vaccination. These results suggest that the thymus-dependent (T) lymphocyte subsets responsible for delayed hypersensitivity, blastogenesis and macrophage activation may be affected differently by protein deprivation. Alternatively, enhanced macrophage activity may not be the result of T cell function in this model.

A new human T-cell differentiation antigen: unexpected expression on chronic lymphocytic leukemia cells.

Monoclonal antibody 10.2 reacts with a monomorphic antigen expressed on the surface of virtually all thymocytes, as well as thymus-dependent lymphocytes in the peripheral blood and bone marrow. In contrast, antibody 10.2 did not react with normal peripheral blood B cells, monocytes, or the non-T-cell fraction of bone marrow. This complement fixing IgG2a antibody also reacted with established leukemic T-cell lines, but not with cell lines of either normal or malignant B-cell origin. Similarly, when tested against acute leukemia blasts, the 10.2 antibody reacted with those from patients with T-cell acute leukemia, but not with those from patients with acute null cell or non-lymphocytic leukemia. An unexpected exception to this pattern was the reaction of 10.2 antibody with leukemic cells from patients with B-cell type chronic lymphocytic leukemia. Immune precipitates formed with 10.2 antibody and detergent lysates of radiolabeled T-cells contained three polypeptides with molecular weights of 65 000, 55 000, and 50 000 daltons. It has not been determined whether all three of these polypeptides contain the 10.2 antigenic determinant, or whether these proteins represent a multimeric antigen complex.

The effect of pre-natal thymectomy on lymphocyte sub-populations in the sheep.

Ten sheep foetuses were thymectomized between 55 and 77 days gestation. The subsequent growth of the lambs was not affected for periods up to 500 days after birth. Prior to 240 days of age the thymectomized lambs were markedly lymphopenic and the response of their blood lymphocytes to phytohaemagglutinin (PHA) was significantly reduced. Peanut agglutinin-binding cells were found to be depleted in the blood of thymectomized lambs, while an unmarked 'null' cell population was virtually absent. The absolute numbers of E-rosette forming cells and sIg+ cells were similar for both groups. These findings indicated that 'null' cells in sheep may be immature thymus-dependent lymphocytes. The effect of thymectomy on blood counts, PHA responsiveness and the numbers of 'null' cells were less evident in thymectomized sheep that survived beyond 240 days. Possible differentiation pathways for sheep T-cells are discussed, together with the role played by the thymus in the maturation of T-cells.

Immunodeficiency in Congenital Chyluria

Chronic thoracic duct drainage in the experimental animal has been employed to deplete thymic dependent lymphocytes with resultant cellular immune deficiency.1-3 The human clinical correlate is the patient with chylothorax or even more analogous, chronic chyluria. The most common cause of chyluria worldwide is filariasis. Other causes include trauma, tuberculosis, neoplasms, infections, lymphactic aneurysm, and thoracic duct stenosis.4 We recently evaluated a child with this defect, giving us the opportunity of evaluating the effect of chronic T-cell depletion on immune reactivity. CASE REPORT J.K. is a 6-year-old Korean boy who was adopted at 2 years of age. Family history is unknown.

Effects of cytotoxic monoclonal antibody specific for T200 glycoprotein on functional lymphoid cell populations

A monoclonal antibody against T200 glycoprotein is selectively cytotoxic for thymocytes and mature thymus-dependent (T) lymphocytes. All T-cell functions assayed, cell-mediated cytotoxicity, helper cell activity, and proliferation in response to T-cell mitogens or allogeneic cells were abolished by prior treatment of spleen cells with anti-T200 antibodies and complement. In contrast, thymus-independent (B) cell responses to lipopolysaccharide (LPS) and other B-cell mitogens were unaffected. Although treatment of spleen cells with anti-T200 antibodies and complement markedly reduced their capacity to mount an in vitro antibody response to sheep red blood cells (SRBC), responsiveness could be restored by the addition of SRBC-primed T-helper cells. Treatment of bone marrow cells with anti-T200 antibodies and complement did not eliminate either in vivo colony-forming units-spleen (CFU-S) or prothymocytes. It is concluded that T lymphocytes become sensitive to complement-mediated lysis by anti-T200 antibodies as a consequence of cell-surface modifications occurring shortly before or just after their entry into the thymus. In contrast to Thy-1 antigen, the selective killing by antibody against T200 glycoprotein cannot be readily accounted for by quantitative differences in the expression of T200 glycoprotein on the cell surface. Fluorescence-activated cell analysis showed that T200 glycoprotein was expressed in similar amount on the majority of all thymocytes, spleen, and bone marrow cells.

Thymosin-dependent T-lymphocyte response in inflammatory bowel disease

Peripheral blood “total” and “avid” thymus-dependent (T) lymphocytes were enumerated in 45 patients with Crohn's disease (CD) and in 23 patients with ulcerative colitis (UC) by using the spontaneous rosette technique (ER). The in vitro effect of thymosin fraction 5, a polypeptide extract of the thymus gland, on avid ER formation was also determined in these patients. The proportion and number of “total” ER were lower in patients with CD (P < 0.02), but not with UC, when compared with controls. More impressive differences were observed when “avid” ER were determined in patients with CD (P < 0.001) and UC (P < 0.05). Incubation with thymosin resulted in a significant increase in “avid” ER in patients with CD and UC, with no such effect observed in the controls. These results indicate that the determination of “avid” rather than “total” ER provides a more sensitive method for detecting alterations in T-cell immune competence. In addition, it is suggested that there is an increased number of circulating T-lymphocytes in CD and UC capable of responding to exogenous thymic factors. This may indicate the presence of a thymosin-responsive immunodeficiency state in these diseases.

Immunotherapy of patients with chronic virus B hepatitis: I. Maturation of human T-lymphocytes under influence of calf thymic hormone

Well known thymic-dependent properties of T-lymphocytes, E-rosetting, and phytomitogenic responsiveness, were observed to be lowered in some patients with chronic virus-B hepatitis. On the assumption that these deficiencies may be due to lack of thymic influence, we decided to attempt their repair by administration of the thymus extract “TFX-Polfa” to the patients. The earliest effect of treatment observed was an increase in percentage of E-rosetting cells to a value within the normal range. Prolonged treatment resulted in the increase of lymphocyte response to stimulation with concanavalin A, and thereafter in significant increase in reactivity to phytohemagglutinin. It is suggested that these quantitative and qualitative improvements elicited by TFX in the peripheral blood lymphocyte pictures of the patients arise out of its ability to induce in vivo sequential processes of differentiation of thymus-dependent lymphocytes normally accomplished by the thymus.

Murine delayed hypersensitivity (DHS): Modulation of in vitro antigen-induced responsiveness of lymph node cells from mice expressing DHS to human γ-globulin by lymphocyte populations from normal syngeneic animals

Using a chemically defined, protein-free medium, the modulatory effect of normal (N) lymphocytes on in vitro antigen-induced proliferation by lymph node cells (LNC) from mice immunized to express delayed hypersensitivity (DHS) to human γ-glogulin (HGG) was quantitated in coculture. LNC from normal syngeneic animals exerted little if any effect on immune-LNC proliferation. Compared with immune-LNC plus N-LNC coculture response. N thymus cells (TC) were consistently suppressive while N spleen cells (SC) varied in their effect from a marginal to a marked potentiation of radiolabeled thymidine incorporation. Inactivation of N-SC suspensions by X irradiation prior to coculture with immune LNC abrogated the increased responsiveness. It therefore appeared that interaction of immune LNC and antigen resulted in recruitment of N-SC to proliferate. Separation of N-SC suspensions to provide enriched populations of thymic-independent (B) and thymic-dependent (T) lymphocytes showed that B cells augmented and T cells suppressed HGG-induced incorporation of [3H] thymidine when cocultured with immune LNC.

Influence of Vitamin E on the Mitogenic Response of Murine Lymphoid Cells

The effect of vitamin E on mitogenesis by polyclonal activators was studied and the vitamin was found to be stimulatory but selective in its action. Vitamin E itself is a mitogen for murine spleen cells. At suboptimal vitamin concentrations, it was capable of stimulating the response to low levels of the thymus-dependent lymphocyte (T cell) mitogen, concanavalin A (conA), but not when conA was itself at optimal levels. When vitamin E was added to the diet at normal levels, it was not as effective in stimulating mitogenesis as it was at much higher levels. The effect of the vitamin on T cell mitogenesis could be modified by the degree of unsaturation of the dietary fat; it was more effective when dietary polyunsaturated fatty acids (PUFA) were low. Under several conditions, it was shown that vitamin E can increase the phytohemagglutinin (PHA)/conA response ratio, which may suggest an effect of the vitamin on the maturation of T cells. In normal mice, vitamin E also stimulated the response to lipopolysaccharide (LPS), a "bursa-equivalent" lymphocyte (B cell) mitogen, but it was unable to do so when spleen cells from athymic, nude mice were used. This suggests a requirement for thymic factors in order for vitamin E to stimulate mitogenesis of B cells.

Functional and molecular organisation of an antigen-specific suppressor factor from a T-cell hybridoma.

Thymus-dependent (T) lymphocytes have been shown to have antigen specificity. The antigen receptor on T lymphocytes, in contrast to that on B lymphocytes, does not appear to be of the conventional immunoglobulin (Ig) type. Studies on the antigen-specific factors derived from helper and suppressor T cells (Ts) demonstrated that they possess determinants with antigen binding affinity and products of genes in the H-2 complex (MHC). Furthermore, antibodies against the variable region of Ig heavy chains or idiotypes have been shown to react with T-cell antigen receptors as well as antigen-specific helper and suppressor T-cell factors (TsF). It is, therefore, conceivable that at least two gene products are involved in the structural entity of these receptors: one each coded for by genes in either. To establish the molecular nature of the recognition component of T cells we have used homogeneous TsF from a T-cell hybridoma with a specific function. We report here that the antigen binding and I-J coded molecules on TsF are independently synthesised in the cytoplasm, and are secreted as an associated form of the two molecules; this association is required for antigen-specific suppression of antibody response.

Stimulation of cytolytic T cells by isolated viral peptides and HN protein coupled to agarose beads.

Sendai virus-infected mouse cells can be lysed by mouse cytolytic thymus-dependent lymphocytes (CTL) directed specifically against the infected cells. The CTL is known to recognise the H-2 antigens on the target cells together with structure(s) including at least the two viral surface glycoproteins also found on purified virus. We report here that anti-Sendai CTL can be stimulated in vitro by detergent-solubilised viral haemagglutinin-neuraminidase (HN), either as the isolated protein or coupled to agarose beads. We further show stimulation by the hydrophilic portion of a protein removed from the virus by the protease subtilisin BPN', and we demonstrate that cyanogen bromide- (CNBr-) cleaved viral peptides also produce such stimulation.

A study of cells present in peripheral lymph of pigs with special reference to a type of cell resembling the Langerhans cell.

Large mononuclear cells with long, actively moving cytoplasmic veils were observed in lymph coming from the skin. The enzyme histochemistry and ultrastructure of these cells suggested that they are related to epidermal Langerhans cells and interdigitating cells in the lymph node. It has been reported that Langerhans cells and interdigitating cells play a role in contact hypersensitivity by taking up antigen and presenting it to thymus-dependent lymphocytes, and it is likely that the veiled cells in the lymph are also involved. After skin-painting with 1-fluoro-2,4-dinitrobenzene (DNFB), the veiled cells in lymph coming from the site of painting became more active and were observed contacting other cells present in the lymph; many large cellular aggregates were found. Since neutrophilic leucocytes and mononuclear phagocytes were the predominating cell types in this lymph, there was no evidence for a massive recruitment of immunocompetent lymphocytes at the site of painting. Neonatally thymectomized pigs do not develop allergic reactivity to DNFB. It is of interest that the number of veiled cells and their ability to form large cellular aggregates was not affected in these animals. Therefore, it is unlikely that the defect in responsiveness can be attributed to a failure in the function of veiled cells.

Antigen-induced in vitro lymphocyte blastogenesis in the rabbit: A T-cell response

In vitro lymphocyte stimulation of sensitized rabbit lymphocytes to specific antigen (ovalbumin) was found to depend on thymic-dependent lymphocytes. This conclusion is based on an enhanced response upon enrichment with T lymphocytes by passage of lymphocytes through nylon wool, and on the elimination of the response after treatment of lymphocytes with complement and an antiserum to rabbit thymus cells prepared in a goat. Specificity of the antiserum was demonstrated by elimination of in vitro T-cell function and retention of in vitro B-cell functions.

Alloantigen Bound to Agarose Beads and Syngeneic Carrier Cells Are Capable of Stimulating Mouse Cytolytic T Lymphocytes in Vitro

Abstract Bead-bound antigen was prepared by coupling alloantigen covalently to agarose beads. Alloantigen-bearing syngeneic carrier cells were prepared by dilution of detergent solubilized alloantigen in the presence of syngeneic spleen cells. Both types of antigen were compared to spleen cells and reconstituted membrane fragments for the ability to stimulate cytolytic thymus-dependent lymphocytes in vitro. All these types of antigen could stimulate immune but not nonimmune spleen cells to form cytolytic T lymphocytes. The amount of lytic activity obtained with the bead-bound antigen was found to be only dependent upon the amount of H-2 antigen present in the culture and independent of the number of beads.

Cell‐Mediated Immunity in Human Pregnancy

The function of thymus-dependent lymphocytes (T lymphocytes) was studied in women during pregnancy and labor and postpartum by evaluating the blastogenesis of peripheral lymphocytes, which were stimulated with phytohemagglutin-P (PHA-P) in both whole-blood semimicroculture and purifed lymphocyte culture. Data from 353 random samples (203 women) and 50 serial specimens from 10 women revealed that PHA-P induced-lymphocyte blastogenesis was significantly (p less than 0.005) reduced during pregnancy and labor but rapidly returned to normal several days after artificial termination in the early stage of pregnancy as well as after full-term delivery. These results indicate that the T-lymphocyte function in maternal peripheral blood is depressed by causes related to pregnancy. It seems very likely that depressed T-lymphocyte function during pregnancy is caused by inhibitory factors in the blood plasma derived from the feto-placental unit. Questions relating to the inhibitory factors in maternal plasma are discussed.

Limited Trypsin Cleavage Distinguishes MHC and Thymus-Leukemia Antigens

A simple technique is presented for the identification of particular cell membrane antigens. The method employs labeled membrane antigens that are isolated immunospecifically and subjected to limited trypsin digestion followed by polyacrylamide gel electrophoresis in detergent. A large "core" peptide is produced by proteolysis of murine thymus-leukemia antigens (TLA) and from antigens of the major histocompatibility complex (MHC). The tryptic cores from H-2K and H-2D are regularly distinguishable from the thymus-leukemia antigens (TLA) by gel electrophoresis in one dimension. This chemical distinction is particularly important in the analysis of antigen mixtures isolated with antisera specific for beta 2 microglobulin. These techniques have been used to identify thymus-restricted beta 2 microglobulin-associated antigens on cell membranes from mouse, man, guinea pig, and monkey. In appropriate inbred mouse strains, these are the TLA and it is proposed that in the three other species examined they may be analogues, although not necessarily homologues, of TLA. The broad species distribution of these thymus-restricted cell membrane antigens suggests that they are involved in the differentiation of thymus-dependent lymphocytes (T cells).

Neutropenia in three patients with rheumatic disorders. Suppression of granulopoiesis by control-sensitive thymus-dependent lymphocytes.

A man with polymyalgia rheumatica (patient 1) and two patients (2 and 3) with Felty's syndrome had neutropenia at the time of diagnosis. Bone marrow samples in each patient were cellular but showed an "arrest" of granulocyte maturation at the myelocyte stage. Agar colony growth of marrow cells from each patient was subnormal but increased after removal of sheep erythrocytes rosette-forming cells (thymus-dependent [T] cells) from marrow cell suspensions before culture. Preincubation of marrow cells with cortisol also enhanced colony growth. Maximum enhancement with cortisol occurred at 1 mM (patient 1), 1 microM (patient 2), and 10 nM (patient 3). Cortisol failed to enhance colony growth after removal of T cells from marrow cell suspensions. Peripheral blood lymphocytes (PBL) and PBL-conditioned medium from all three patients inhibited colony growth of normal human marrow cells. Cortisol treatment of PBL or T depletion from PBL abrogated the inhibition in coculture and with conditioned medium. Prednisone therapy resulted in the disappearance of suppressor T-cell function concomitant with hematologic improvement in patients 2 and 3, but suppressor T cells persisted in patient 1, who did not respond to prednisone. We conclude that cortisol-sensitive T lymphocytes inhibited granulopoiesis in vitro probably by elaboration of a soluble factor or factors. Our results suggest (a) that neutropenia in these patients resulted, at least in part, from T-cell suppression of granulopoiesis, (b) that the effectiveness of prednisone therapy was a result of its inhibition of suppressor T cells, and (c) that responses to glucocorticoid therapy may be predicted in such patients with the agar culture technique and cortisol dose response in vitro.

E-rosette receptors induced by phytohemagglutinin on human K cells expressing T-cell surface antigens

Killer cells (K cells) enriched from human blood mononuclear cells which mediate antibody-dependent cellular cytotoxicity (ADCC) were examined for surface markers. Sixty-seven percent of the E-rosette-negative, sIg-negative cells reacted with anti-T cell serum (AMT) previously shown to react with immunochemically defined T-cell antigens. Phytohemagglutinin induced 25% of K cells to express an E-rosette receptor. When these induced cells were isolated, greater than 98% reacted with AMT and 17% expressed the Fc receptor for IgG. Furthermore, they retained their functional capacity in ADCC. These findings demonstrate that an E-rosette receptor can be induced on human K cells. The data suggest the K-cell fraction included a population of thymus-dependent lymphocytes which can function as effector cells in ADCC.

In vitro thymosin effect on T lymphocytes in alcoholic liver disease

The in vitro effect of thymosin fraction 5, a thymic gland extract, on thymus-dependent lymphocytes was studied in 12 patients with alcoholic hepatitis (AH) and in 18 patients with compensated alcoholic cirrhosis (CAC). The baseline number and proportion of spontaneous rosette-forming T lymphocytes were significantly reduced in AH as compared to the controls. No such decrease was seen in CAC. When the lymphocytes of patients with AH were incubated in the presence of thymosin fraction 5, the subsequent number and proportion of rosette-forming T lymphocytes showed a significant increase. There was no significant reponse to thymosin in CAC or in control subjects. There appears to be a subset of immature T lymphocytes in patients with AH that is responsive to exogenous thymic factors with subsequent activation of the capacity to form spontaneous rosettes.

Thymosin immunotherapy in patients with small cell carcinoma of the lung. Correlation ofin vitro studies with clinical course

Previous studies suggest that the mechanism of action of thymosin is reconstitution of immune defects rather than augmentation of relatively normal levels of cellular immunity. To test this hypothesis, we correlated in vitro assays with clinical course in 55 patients with bronchogenic carcinoma who were randomized to receive subcutaneous inoculations of thymosin 60 mg/m2 (T60), thymosin 20 mg/m2 (T20), or no thymosin (P) twice weekly during the first six weeks of an intensive chemotherapy program. Overall tumor response did not differ significantly among the three treatment groups (p >.12); however, survival of the T60 group was significantly greater than that of the P group (p =.017). Duration of response for patients achieving a complete remission was significantly longer in the T60 group than in the P group (p =.02). In an attempt to define an association between the increased survival of the T60 group and in vitro assays that show changes with tumor presence and extent, comparisons were made between survival and pretreatment peripheral blood levels of carcinoembryonic antigen (CEA), α2HS-glycoprotein (α2HS), and total thymus-dependent lymphocytes (T cells). Independent of therapy, pretreatment CEA levels correlated directly with tumor extent (p=.004) and inversely with degree of tumor response (p=.006) and survival (p=.006). The effect of T60 compared with the P group on survival was similar in patients with pretreatment CEA levels >4 Ng/ml and those with levels >4 Ng/ml. However, among those with pretreatment total T-cell and α2HS levels below the median for all patients, survival of the T60 group was significantly greater than that of the P group (p=.006 and p=.036, respectively); for patients with higher pretreatment total T-cell and α2HS levels, survival did not differ significantly between the T60 and P groups (p=.28 and p=.17, respectively). Analysis by tests of interaction shows that the effect of T60 on survival depended on pretreatment levels of α2HS (p=.046) and, to some extent, total T-cells (p=.17). Thus the association between survival and type of treatment may be related to pretreatment levels of these two parameters of cellular immunity. These results support the hypothesis that patients with relatively low levels of immunity benefit most from administration of thymosin. Additional studies with larger patient populations and other tumors are needed to confirm these findings. Cancer 43:863–870, 1979.