Heterologous Antilymphocyte Serum Research Articles

A semi-automated technique for the isolation of live lymphocyte suspensions from peripheral blood.

Abstract A semi-automated method has been devised for the separation of viable human peripheral blood lymphocytes from standard units of donor blood. The tech- nique is based on the carbonyl iron method (Levine, 1956; Cassen, Hitt, and Hays, 1958), but using a continuous-flow system. Lymphocytes harvested by this technique have been used in the production of heterologous antilymphocytic serum (ALS).

Cell migration and chimerism after whole-organ transplantation: The basis of graft acceptance

Improvements in the prevention or control of rejection of the kidney and liver have been largely interchangeable (1, 2) and then applicable, with very little modification, to thoracic and other organs. However, the mechanism by which anti rejection treatment permits any of these grafts to be “accepted” has been an immunological enigma (3, 4). We have proposed recently that the exchange of migratory leukocytes between the transplant and the recipient with consequent long-term cellular chimerism in both is the basis for acceptance of all whole-organ allografts and xenografts (5). Although such chimerism was demonstrated only a few months ago, the observations have increased our insight into transplantation immunology and have encouraged the development of alternative therapeutic strategies (6).

Cell migration and chimerism after whole-organ transplantation: The basis of graft acceptance

Cell migration and chimerism after whole-organ transplantation: The basis of graft acceptance

Suppression of skin allograft rejection by post-transplantation administration of specific anti-lymphocyte serum

An heterologous anti-lymphocyte serum ALS(I-GR), was raised in rabbits by immunization with draining lymph node cells of AKR mice which had rejected DBA/2 skin allografts. Treatment of AKR mice with this ALS on the 4th day after DBA/2 skin grafting, significantly prolonged the survival of the graft in comparison with that in allografted mice treated with normal rabbit serum. In contrast, ALS prepared against unsensitized lymph node cells was found to be ineffective when administered after transplantation. A further prolongation of allograft survival was obtained when ALS(I-GR) was administered to recipients on days +4 and +7. ALS(I-GR) seemed to specifically suppress the rejection of DBA/2, but not of C57 BL/6 skin grafts. The suppressive action of ALS(I-GR) was not due to cross reactive (anti-DBA) antibodies and was probably directed against idiotypic determinants on antigen-stimulated cells.

Suppression of antibody response to transfusions in rats by preconditioning with antibody-coated cells.

In a Lewis-BN rat model, the antibody response to transfused blood cells from strongly histoincompatible donors was suppressed by preincubation of blood with specific antidonor antiserum or broadly reactive heterologous antilymphocyte serum (ALS). Suppression was achieved even when excess antiserum was removed by washing prior to injection. The suppressive effect was dose-dependent. Broadly reactive ALS was even more effective than specific antidonor antiserum in inhibiting the primary antibody response. Lewis animals pretreated three times with ALS-coated BN blood showed no secondary antibody response against subsequent BN-blood booster injections. Our experiments may be relevant for the prevention of undesired sensitization to donor-specific transfusions prior to related donor kidney transplantation.

In vitro studies on the effect of anti-lymphocyte serum on the humoral immune response.

The effect of ALS (I), a heterologous anti-lymphocyte serum prepared against lymph node cells from rats pre-immunised with sheep erythrocytes (SRBC), on plaque forming cells (PFC) to SRBC was studied in vitro. ALS (I) reduced the number of both IgM and IgG PFC when complement was included in the reaction. This ability of ALS (I) to inhibit PFCs in vitro was absorbed out by the IgG fraction of anti-SRBC serum. Thus ALS (I) was thought to possess and anti-idiotypic antibody directed against B-cells at a later stage of differentiation.

Glycoproteins of murine thymocyte and splenocyte surface membranes; binding to Concanavalin A and recognition by heterologous antilymphocyte serum

Surface proteins of murine thymocytes and splenocytes were radio-iodinated by a lactoperoxidase-catalyzed reaction and solubilized using the nonionic detergent Nonidet P-40. Labelled glycoproteins which bound to insolubilized Concanavalin A were eluted specifically with 200 m M α- methyl- d-mannopyranoside and analysed by polyacrylamide gel electrophoresis in sodium dodecylsulfate. Polyacrylamide gel electrophoresis resolved Con A binding glycoproteins of thymocytes and splenocytes into 6 and 9 distinguishable components, respectively. Although there was a general similarity between the Con A binding components some clear differences were found. Most notably, thymocytes possessed a prominent surface glycoprotein of apparent mass 22–27,000 daltons which was absent from the spelnocytes. In addition were enriched in components of apparent mo. wt 40–45,000 and 65,000 relative to thymocytes. All radio-iodinated lymphocyte surface proteins recognized by a horse anti-mouse thymocyte serum bound to Con A-Sepharose. This antilymphocyte serum was shown to recognize predominantly one (or a small number) of high mol. wt ( > 150,000 daltons) glycoproteins present on both thymocyte and splenocyte membranes.

Glycoproteins of murine thymocyte and splenocyte surface membranes; binding to Concanavalin A and recognition by heterologous antilymphocyte serum

Glycoproteins of murine thymocyte and splenocyte surface membranes; binding to Concanavalin A and recognition by heterologous antilymphocyte serum

Dark reticular epithelial cells of the thymus as the primary target of heterologous anti-lymphocyte serum in [formula omitted] mice : An ultrastructural study correlated with data on thymus substitution

Effects of heterologous anti-lymphocyte serum (ALS) administration on the ultrastructure of thymus have been investigated in intact and adrenalectomized BALB c mice. The alterations of dark reticular epithelial cells (DREC) were seen already at 1 hr after serum administration to intact mice. A large portion of DREC disappeared from the thymus. Adrenalectomized mice showed basically the same ultrastructural pattern of alterations, their degree, however, and incidence being lower than those of intact mice. The alterations of thymocytes occurred later than those of DREC. In parallel experiments with thymus substitution it was shown that, in contrast to normal untreated thymuses, ALS-treated thymuses are not able to contribute to the overall recovery from ALS effects, when installed in thymectomized recipient mice. The recovery of normal DREC ultrastructure began in both experimental groups of animals at about 24 hr after serum administration. It was concluded that DREC damage is provoked by several factors one of which might be the direct action of ALS on DREC. A model of the structural and functional involvement of the thymus as a consequence of ALS administration is proposed.

Effects of Concanavalin A on the migration of radioactively labeled lymphoid cells

The effects of the jackbean globulin Concanaalin A (Con A) on the distribution of radioactive 51Cr-labeled lymph node cells was studied in CBA mice. Lymph node cells treated in vitro with Con A in subagglutinating noncytotoxic doses were unable to “home” to the lymph nodes of syngeneic recipients after intraenous injection. The effect was almost immediate and seemed unrelated to mitogenesis. The inhibitory effect of Con A on lymphocyte migration could be partially reersed by alpha-methyl mannoside; the degree of migratory impairment was related to the amount of Con A bound to the lymphocyte surface at the time of transfer. The membrane site at which Con A binds to the lymphocytes is similar to that which is bound by heterologous antilymphocyte serum but is probably distinct from the theta antigenic site. These data lend support to the hypothesis that surface lymphocyte carbohydrate determinants are involved in the specific lymphocyte “homing” receptor.

Immunity to Hymenolepis citelli by Peromyscus maniculatus: Genetic Control and Ecological Implications

Acquired resistance to Hymenolepis citelli in Peromyscus maniculatus, the white-footed deer mouse, is shown to be controlled by a single autosomal dominant gene R (recessive gene designated r). This resistance could be transferred to uninfected hosts with lymphoid cells but not serum from infected resistant animals. Treatment with heterologous antilymphocyte serum depressed the ability of competent hosts to resist infections. A comparison of the response elicited against 2 strains of H. citelli (Utah and California) illustrated that chance-bred P. m. sonoriensis respond more rapidly and in greater incidence to the California strain than to the Utah strain. Homozygous recessive (r/r) hosts, susceptible to H. citelli (Utah), respond in significant numbers to the California strain of the tapeworm. Natural infections of H. citelli in Peromyscus do not occur uniformly throughout the environment but occur in small foci favoring existence of both intermediate and definitive hosts. Infections rarely occur outside such foci. We postulate that H. citelli is propagated in such foci by genetically nonimmune (r/r) hosts comprising about 25% of the host population. A previous study (Wassom et al., 1973), utilizing the tapeworm Hymenolepis citelli and its natural host Peromyscus maniculatus, illustrated that a tapeworm living exclusively in the gut lumen could elicit a protective resistance in some individuals. Such hosts, after initial acceptance of infection, responded by eliminating the worms and resisting reinfection. Other individuals were incapable of developing resistance. Primary infections were maintained and secondary infections could be imposed upon initial ones. The present study was conducted to evaluate the genetic aspects of this host resistance, and to determine whether the response is mediated by specific immune mechanisms or by nonspecific factors. MATERIALS AND METHODS All experimental animals were laboratory-reared Peromyscus maniculatus sonoriensis fed Purina Laboratory Chow. The isolation of H. citelli from wild-caught P. maniculatus and its maintenance in the laboratory as well as procedures for infection have been previously reported (Wassom et al., 1973). Immune serum was pooled from 2 groups of chance-bred P. maniculatus infected on day 0 with 10 cysticercoids of H. citelli, challenged with reinfection on day 14 or 21, and bled 7 days later. Received for publication 16 July 1973. * Department of Biology, University of Utah, Salt Lake City, Utah 84112. t Department of Pathology, University of Utah, Salt Lake City, Utah 84112. 47 Lymphoid-cell suspensions from the thymus, spleen, and lymph nodes of infected animals were prepared by mincing the tissues in Minimal Essential Medium for Suspension-Earle's BSS (MEMMicrobiological Associates), filtering through nylon gauze, and centrifuging at 60 g for 10 min. Red blood cells were lysed with Tris-buffered hypotonic ammonium chloride (Boyle, 1968), and the cells washed 4 times in MEM. Viability of cells was determined by trypan blue exclusion (Boyse et al., 1962). Cell concentrations were determined with standard microscopic counting chambers. Antilymphocyte serum (ALS) was prepared in rabbits by injection of Mus musculus lymphoid cell membranes in Freund's complete adjuvant. Injection of 0.5 ml of this ALS intraperitoneally on days 0, 2, and 4 was shown to delay median rejection times of Mus musculus allogeneic skin grafts (H2' to H2k) from 10.5 to 13.7 days. Skin grafts were not attempted in P. maniculatus. However, injection of 0.4 ml ALS intraperitoneally to P. maniculatus on days 0, 2, and 4 was shown to reduce hemagglutinin titers to SRBC by 4 doubling dilutions. This ALS is thus an effective immunosuppressive agent in P. maniculatus.

Cellular and humoral immunity after allogeneic transplantation in the rat. IV. The effect of heterologous antilymphocyte serum on cellular and humoral immunity after allogeneic renal transplantation.

Cellular and humoral immunity was measured in Lewis rats that received (Lewis X Brown Norway) F1 (L X BN) renal allografts with or without rabbit anti-Lewis lymphocyte serum (ALS). A 51Cr release assay and a microcytotoxicity assay were used to measure cellular immunity (CMI) of immune lymphoid cells at various times after transplantation. As measured by the 51Cr release assay, ALS markedly suppressed the development of CMI; this virtual absence of CMI correlated well with the lack of immunological damage to renal allografts as measured by low levels of blood urea nitrogen (BUN) and prolonged allograft survival. As measured by the microcytotoxicity assay, the development of CMI was slightly delayed by ALS and had no relationship to levels of BUN. Also, following transplantation and ALS treatment, serum antibody production was almost completely suppressed. The disparate courses of CMI, as measured by the 51Cr release and microcytotoxicity assays, may be attributable to measurement of different populations of effector cells in each or to the measurement of different states of cellular immune activity in vitro. The mechanism by which ALS causes indefinite renal allograft survival is not clear. As measured by the 51Cr release assay, ALS can inhibit the cytotoxic effect of immune lymphoid cells in vitro in the absence of complement. Rats given ALS at various times prior to renal transplantation gradually recovered the ability to develop CMI as the interval between ALS pretreatment and transplantation was lengthened. Significant recovery of the ability to develop CMI was evident by 50 days after ALS pretreatment, and full recovery had occurred by 100 days. In contrast, all allograft recipients given ALS at the time of transplantation survived more than 100 days with relatively normal levels of BUN. The mechanism by which ALS causes indefinite renal allograft survival may be suppression of CMI for sufficient time to allow production in vivo of blocking serum factors that inhibit CMI and prevent rejection. Immunological damage to the allograft is then determined by the continued suppression of CMI by the blocking serum factors.

Reversal by the MER tubercle bacillus fraction of the suppressive effects of heterologous antilymphocytic serum (ALS) on the allograft reactivity of mice

Treatment of immunized C57B1 mice with the MER fraction of BCG prevented wholly or in large part the strong immunosuppressive effects elicited by heterologous ALS against allograft reactivity, as measured by the in vitro cytotoxic activity of spleen cells against the immunizing allogeneic cells (a BALB/c plasmacytoma). Moreover, the heightened cytotoxic capacity of spleen cells bestowed by treatment of the donor with MER was manifested in some instances despite exposure to ALS. Spleen cells from MER-treated hosts were also more refractory to the direct cytotoxic action of ALS and complement in vitro.

Heterologous antilymphocyte serum hastens the growth of 7,12-dimethylbenz(alpha)anthracene induced tumours in mice.

The present paper describes the effects of repeated administration of rabbit anti-mouse lymphocyte serum (ALS) or normal rabbit serum (NRS) on tumours induced in Charles-River mice by 7,12-dimethylbenz(α)anthracene (DMBA) given at birth. ALS or NRS were given either at the time of DMBA administration and subsequently at weekly intervals for the first 10 weeks of life, or at daily intervals for 7 days during the first, second, third or fourth week of life. The incidence and histology of the tumours were studied. It was found that treatment of very young mice with ALS greatly reduced the mean survival time of mice and significantly increased the incidence of malignant lymphoma. The incidence of lung tumours was found to be significantly increased in the animals injected with ALS during the second week of life.Treatment with ALS in the other experimental groups gave results essentially similar to those observed in DMBA control and NRS treated mice.

Diffusible factor of thymus is responsible for the recovery from some effects of heterologous antilymphocyte serum.

Nel sangue di topi timectomizzati trattati con ALS un alto e prolungato livello di FALA si accompagna ad una linfopenia mentre nei topi non-timectomizzati il pronto ripristino dei valori leucocitari si attua in concomitanza alla rapida scomparsa dei FALA dal siero. Viene messo in evidenza che responsabile del ripristino di questi effetti del siero antilinfocitario e un fattore umorale diffusibile del timo.

Time course and mechanism by which alloantiserum raised against thoracic duct lymphocytes abrogates runt disease in the rat.

SUMMARY An alloantiserum (AAS) prepared in adult Brown Norway (BN) rats against Lewis (L) thoracic duct lymphocytes (TDL) exerted anti-L lymphocytotoxins and lymphoagglutinins in vitro and was capable of aborting an already developed graft-versus-host (GVH) reaction caused by L-TDL in neonatal BN rats in vivo. A blastogenic activity was not provoked by the antiserum in L lymphocytes either in vitro or in vivo. However, the AAS did promote the disappearance of L lymphocytes transfused into adult and neonatal BN animals with a higher efficiency in the adult. These observations demonstrated that the AAS did not act solely on circulating lymphocytes but also on those L cells resident in BN lymphoid tissue, because they were preferentially removed in BN animals containing “homed” radioactively labeled L lymphoid cells by lymph node reticular cells. In contrast, BN animals given antiserum prior to being transfused with L cells, and hence possessing circulating antibodies specific for the labeled allogeneic L inoculum, removed the lymphocytes very rapidly (within 2 hr or less), chiefly by action of phagocytic blood sinusoidal lining cells. Therefore, specific removal seemed to occur in animals given antiserum by whatever BN phagocytic cell the L lymphocyte had first contacted. Moreover, removal of circulating and homed cells within 24 hr in the adult and at least 36 hr in the neonatal BN animal, suggested that the antiserum penetrated into lymphoid tissue. This is in contrast to heterologous antilymphocyte serum (ALS) which had been reported not to penetrate into tissues. As transfused radioactively labeled L lymphocytes contained small cells of both newly formed and longer lived varieties, and because all labeled cells were removed by the antiserum, the AAS did not appear to affect one population of small lymphocytes in preference to another.

Studies on heterologous antilymphocyte serum in mice. IX. In vitro assay by indirect leukoagglutination.

Studies on heterologous antilymphocyte serum in mice. IX. In vitro assay by indirect leukoagglutination.

Studies on the isolation of lymphocytes active in cell-mediated immune responses: III. The pattern of recovery of immuno-competence by various lymphoid cell populations following cessation of chronic administration of antilymphocyte serum

Long-term four month treatment with heterologous antilymphocyte serum (ALS) produced immunoincompetence of thymus, spleen and bone marrow lymphocytes as measured by their ability to produce a graft-versus-host reaction. Twenty-four hours following discontinuance of treatment this immunoincompetence is manifested by all three cell populations. Forty-eight hours following cessation of administration of ALS an immunocompetent population of lymphoid cells is demonstrable in the thymus gland. This activity is again confirmed at 168 hr, a time at which no immunocompetent cells are detectable in the spleen or bone marrow cell populations. This pattern of recovery of immunocompetence by populations of lymphoid cells helps to (1) localize the primary site of immunoconversion of stem cells into immunocompetent cells in the thymus gland; (2) serves to confirm the primary action of ALS as one of destruction of immunocompetent cells and to infer that its action is not to interfere with the thymus-guided maturation processes; (3) defines the recovery phase following ALS treatment as that time required for the build up of a population of immuno-competent cells in the thymus with subsequent seeding of these cells to the peripheral lymphoid organs; and (4) confirms that the normal thymus contains a population of immunocompetent cells whose activity can not be ascribed to contaminating blood or lymph node lymphocytes.

Studies on the isolation of lymphocytes active in cell-mediated immune responses: II. Identification and recovery of an immunocompetent subpopulation of mouse thymocytes

Suspensions of mouse thymocytes were separated into subpopulations of cells by centrifugation on discontinuous bovine serum albumin (BSA) gradients. Nine bands or fractions of cells localized at the interfaces of the ten different concentrations of albumin used. The fractions were numbered from 1 through 9, beginning with least dense harvested at the 17/19% interface and thereafter counted sequentially to the most dense. Fraction 5 comprised at least 50% of the cell population while fractions 1–4 constituted approximately 20% of which 15–18% were in fraction 4. Each fraction was then evaluated individually to assess its reactivity. Five parameters of responsiveness were used: 1. Incorporation of tritiated thymidine in tissue culture and in vivo; 2. Response to stimulation with the mitogen phytohemagglutinin ; 3. Homing or migratory potential to peripheral lymph nodes; 4. Ability to provoke a significant graft-versus-host reaction; and 5. Sensitivity to heterologous antilymphocyte serum. Fractions 2, 3 and 4 each possessed at least one of these attributes of thymus derived small lymphocytes, although others were lacking. Fraction 5 was inert in all systems tested. The striking depletion of bands 2–4 by ALS was reversible by the injection of marrow precursor cells obtained by differential centrifugation of syngenic marrow. Marrow cells from fractions 6–10 of marrow gradients did not replenish the depleted thymus. In whole organ perfusion culture fractions 2–4 were found to egress from the gland while the bulk of cells remained behind. It was concluded that there is a small active immunocompetent population of thymocytes within the thymus which undergoes sequential maturation and/or modulation, so that some surface changes may be necessary for circulation but not for competence.

Effect of Heterologous Antilymphocyte Serum on the Spontaneous Cure of Trichinella spiralis Infections in Mice

Effect of Heterologous Antilymphocyte Serum on the Spontaneous Cure of Trichinella spiralis Infections in Mice