Normal Lymphocyte Transfer Research Articles

Biphasic response of the regional lymphatics in the normal lymphocyte transfer reaction.

Initially developed for histocompatibility testing, the normal lymphocyte transfer (NLT) reaction involves the intradermal injection of allogeneic lymphocytes from one individual to another. Because of the unique kinetics of the immunological response to allogeneic lymphocytes, the NLT reaction has been considered an informative system for the analysis of transplant immunity. In this study, we used bilateral efferent lymph duct cannulations in sheep to examine the regional lymphatic response to the NLT reaction. Our studies used monoclonal antibodies to define lymphocyte population dynamics and DNA flow cytometry to reflect lymphocyte proliferative responses. The results confirmed a biphasic NLT reaction. An unexpected finding was the marked differences between the early and late NLT responses. The early response was characterized by T-lymphocyte proliferation, as reflected by S-phase DNA, which was comparable in both the NLT-stimulated and contralateral control efferent lymphocytes. This bilateral proliferative response was observed in both CD4+ and CD8+ lymphocytes. In contrast, the late response was restricted to the efferent lymph from the NLT-stimulated lymph node. Dual-parameter flow cytometry demonstrated that the dominant component of this unilateral NLT response was CD8+ lymphocytes. These results suggest important functional distinctions between systemic and regional lymphatic responses to intradermal alloantigens.

Progress in the determination of histocompatibility. 1965.

Experimental and clinical data currently available suggest that of the various possible ways in which the outcome of organ homotransplantation might be improved upon, the one which holds most immediate promise involves devising a selection procedure capable of excluding gross histoincompatibilities. The various approaches to the problem of tissue typing or matching that are presently being investigated are reviewed from the viewpoint of their underlying principles and their potentialities. These methods include direct grafting tests, serologic procedures aimed at matching or identifying pertinent isoantigenic specificities associated with formed elements of the blood, especially leukocytes, and various extracorporeal biologic assays of the capacity of an intended host's immunologically competent cells to react against the transplantation isoantigens of a possible donor. The latter category of tests includes the normallymphocyte transfer test and the irradiated-hamster test.

Endocytic stripping of ligands from migrant lymphocytes in high endothelial venules (HEV): implications for immunomodulation vs viral pathogenesis.

“Grooming ” of membrane detritis from lymphocyte surfaces by mononuclear phagocytes during migration through lymphatic tissues was proposed as a cause of differences in the potency of comparable populations of lymphocytes to produce the normal lymphocyte transfer reaction (LTR) when equivalent numbers of T cells from blood, afferent lymph, and efferent lymph were injected SC1. Three- to five-fold greater LTR activity was found with efferent lymph cells compared to blood or afferent lymph cells. It is possible that these differences in reactivity were due to anti-idiotypic antibodies or other “blocking factors ” occupying lymphocyte receptors. Isotypic, allotypic and idiotypic immunoregulation occurs in mice, rats, rabbits, and man2•5. The present studies employ light and electron microscopy, cytochemistry and immunochemistry to analyze the endocytic potential of Lewis Rat HEV endothelium toward both particulates and toward autologous lymphocytes whose surfaces have been modified by liganding. Results indicate that HEV endothelial cells nonspecifically remove receptor-blocking proteins during blood to tissue transit.KeywordsAlcian BlueHigh Endothelial VenuleMigrate LymphocyteLymphocyte SurfaceThoracic Duct LymphThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Fluorescein isothiocyanate as a cell marker for tracing lymphocyte circulation in sheep.

The use of fluorescein isothiocyanate (FITC) as a cell marker in ovine lymphocyte circulation studies was investigated. The effects of the label on lymphocyte function were assessed by lymphocyte blastogenesis, cell-mediated cytotoxicity and normal lymphocyte transfer reactions. Labelling lymphocytes in 0.05 mg FITC/ml for 10 min at 37 degrees C was found to be optimum. Circulation kinetics of FITC-labelled efferent lymph lymphocytes were monitored as they disappeared from peripheral blood following intravenous infusion and reappeared in the efferent lymphatic duct of the popliteal lymph node. No functional changes were detected in lymphocytes labelled with the optimal regimen.

Immunological reactivity between chimeric cattle twins. III. Mixed leukocyte reaction.

Leukocytes from chimeric calves invariably failed to mount a proliferative response in excess of control responses when cultured with cotwin cells as stimulators. The levels of cellular proliferation in control cultures of leukocytes from chimeric donors were so low as to exclude the occurrence of any auto-stimulation, and it was clear that the absence of excess proliferation in mixed cultures could not be attributed to elevated background levels. Attempts to restore reactivity in mixed leukocyte culture by removal of cell subpopulations from chimeric calf leukocytes and by modification of cell surfaces were unsuccessful. It was inferred that the unreactivity between chimeric cotwins manifest in the normal lymphocyte transfer and mixed leukocyte reactions was unlikely to be produced by humoral or cellular blocking mechanisms.

Genetic control of allogeneic interactions in the guinea pig. I. Evidence from mixed leukocyte culture and normal lymphocyte transfer reactions.

AbstractThe role of gene products of the B region of the guinea pig major hist ocompatibility complex (MHC) in the production of mixed leukocyte culture mitogenic factor (MLC‐MF) has been investigated. Lymphoid cells of partially inbred and outbred guinea pigs which bear the serologically defined B.2 alloantigen, respond poorly or not at all in the guinea pig MLC. Since MLC culture supernatants from B.2+ cells do not stimulate B.2− third party lymphoid cells, it is possible that either B.2+ lymphoid cells do not elaborate MLC‐MF or they produce a suppressor factor. Genetic studies have demonstrated that genes closely linked to or identical with the gene(s) coding for the B.2 alloantigen, regulate “MLC unresponsiveness”. By contrast to their unresponsiveness in the MLC, B.2+ lymphoid cells produce migration inhibition factor at least as well as cells bearing the B.1 and B.3 specificities, and they respond in vitro to antigens which are under the control of specific immune response genes.The genetic restrictions for responsiveness in the MLC are supported by in vivo studies which strongly suggest that the degree of interaction between allogeneic lymphoid cells (normal lymphocyte transfer, immune lymphocyte transfer and direct reactions) are also under the control of genes which map in or near the B region of the guinea pig MHC. Preliminary evidence suggests that the B.2+ donor cells rather than B.2+ host cells are responsible for initiating the unresponsiveness associated with the normal lymphocyte transfer reaction. These studies should provide a basis for the reproducible production of MLC‐MF and a system for the study of some molecular aspects of allogeneic interactions in vitro and in vivo.

Normal lymphocyte transfer reaction in humans and its possible mechanism.

This study illustrates the development of the skin reaction in human recipients to injected allogeneic lymphocytes from two aspects: the capacity of lymphocytes to elicit the reaction, and the ability of the recipient to react. The reaction is expressed by local skin induration and is biphasic. The first phase seems to be a hostgraft reaction; the second may require proliferation of graft cells as well as the reaction of host cells. Circulating fetal lymphocytes collected from a 12-week human embryo caused no reaction in an adult recipient; those from a 15-week embryo usually caused the first phase, with little or no second phase reaction, while those from 22- and 30-week fetuses and from a mature infant caused a normal, biphasic reaction. So, usually, did 22-week splenic lymphocytes, but fetal thymic lymphocytes usually provoked no reaction in adults. In premature infants given adult cells either both phases or the second were reduced; in normal infants the second response may be reduced but after six years of age the reaction is like that seen in adults.

The Role of Hyperemia in Cellular Hypersensitivity Reactions

The three physiological processes vascular permeability, blood flow and lymphocyte migration were all enhanced in tuberculin reactions induced in guinea pigs and sheep and also in normal lymphocyte transfer reactions in sheep. Microspheres labelled with 85Sr were used to measure blood flow to dermal sites and it was found that cellular hypersensitivity reactions had blood flows 7-25 times that of normal skin at the reaction peak. Vascular permeability was measured as an increase in the flow rate of afferent lymph or, in guinea pigs, as the enhanced leakage of intravascular 125I-albumin. When the permeability-inducing peptide bradykinin was injected directly into tuberculin reaction, the resulting permeability was greater than the sum of the tuberculin and bradykinin permeability taken individually and it was concluded that the hyperemia enhanced the permeability-inducing capacity of bradykinin. When the traffic of lymphocytes through hypersensitivity lesions was measured in sheep by cannulating the regional afferent lymph vessels and continuously collecting the lymph, the increase in lymphocyte traffic was of the same order of magnitude as the increase in blood flow. It is suggested than the antigen-induced enhancement of blood flow caused the increase in lymphocyte traffic and that the mechanism was similar to that occurring within lymph nodes during the immune response to all antigens.

Normal Lymphocyte Transfer Reaction in Humans and its Possible Mechanism

Normal Lymphocyte Transfer Reaction in Humans and its Possible Mechanism

Guinea Pig Histocompatibility Antigens: III. Analysis of Normal Lymphocyte Transfer (NLT) Reactions among Guinea Pigs with Serologically Defined Phenotypes (GPL-AB Locus and I Region)

The normal lymphocyte transfer (NLT) reaction occurring upon intradermal injection of lymphocytes into a normal recipient may be considered as a kind of mixed lymphocyte reaction in vivo. In previous experiments, we have defined serologically a number of guinea pig lymphocyte antigens, controlled either by a GPL-A locus (equivalent of H-2 D locus in mice) or by a so-called I region (equivalent to the I region in mice). The I region also appears to control a number of immune response genes in the guinea pig.Using guinea pig inbred strains (such as 2, 13, OM3, BE), guinea pig families homozygous for their GPL-A antigens and serologically characterized outbreds, it has been possible to show that antigens of the I region probably play a major role in NLT reactions. No NLT reactions occur among outbred animals of a closed colony which have been bred to homozygozity for their GPL-A antigens and thereby probably possess haplotype homozygozity for their major histocompatibility complex. The NLT reaction among serologically characterized guinea pigs may become a convenient way to detect new specificities and recombinant progeny.

Studies on the lymphocytes of sheep. II. Some properties of cells in various compartments of the recirculating lymphocyte pool.

The proportion of cells bearing surface immunoglobulin (Ig) and the normal lymphocyte transfer (NLT) reactivity of mononuclear cells obtained from the blood, afferent lymph, and efferent lymph from both somatic and intestinal nodes of sheep were measured. The proportion of Ig-positive lymphocytes was approximately 30% in blood and efferent lymph, but only about 10% in afferent lymph. The proportion of unlabeled lymphocytes (T cells) was about the same in all the preparations, as afferent lymph contains 15--20% macrophages. Despite this apparent uniformity in the T cell content, the NLT reactivity of efferent lymph was 3 to 5 times higher than that of blood or afferent lymph. No humoral factors could be detected by incubating cells in the various plasmas in vitro before injection. No inhibitory cells could be detected when mixtures of cell populations were used to initiate lesions. We suggest that lymphocytes in efferent lymph are more reactive than others because they are "groomed" by macrophages during their recirculatory passage through the substance of the node.

3H-Uridine Incorporation by Small Lymphocytes of Tolerant Rats: Relationship to T and B Lymphocytes

Tissue tolerance was induced in neonatal rats by the intravenous injection of bone marrow cells from adult allogeneic rat donors. After 6 to 8 weeks, lymphoid cells from rats in which tolerance had been induced were tested for mixed lymphocyte reactivity (MLR), 3H-uridine uptake, and the relationship of uridine incorporation to B and T lymphocytes. Lymph node (LN) and spleen (SPL) cells from the adult inoculated rats showed no reactivity in the MLR or normal lymphocyte transfer reaction (NLTRx), indicating that the animals were tolerant. After in vitro exposure to 3H-uridine, an abundance of small lymphocytes (SL) from these same tolerant rats were heavily labeled, in contrast to nontolerant controls, where relatively few SL were heavily labeled. In order to determine whether the heavily uridine-labeled cells were T cells or B cells, lymphoid cells from the LN and SPL of tolerant animals were exposed to either rabbit anti-AKR brain serum or rabbit anti-rat Ig conjugated with ferritin. The results showed that the heavily uridine-labeled SL of the tolerant rats were mainly Ig-positive cells.

Lymphocyte reactivity in patients with carcinoma of the breast and large bowel.

The reactivity of lymphocytes from patients with either carcinoma of the breast or large bowel has been studied using the human to mouse normal lymphocyte transfer (NLT) reaction. It was found that, in the case of breast cancer, there was a direct correlation between the clinical stage and a reduced NLT reaction. Only patients with regional lymph node or generalized metastases showed significantly reduced lymphocyte reactivity. However, in the case of large bowel cancer there was a generalized reduction in NLT reactivity which was independent of the clinical stage. Incubation of lymphocytes from individuals without neoplastic disease in serum or plasma from breast cancer patients, showing reduced NLT reactivity, resulted in a reduced NLT reaction. This appears to be indicative of the presence of circulating "blocking factor" in such patients.

Transplantation of lymphocytes and immune lymphocytes in sarcoidosis and in malignant lymphadenomas.

Summary When lymphocytes of a homologous donor are transferred intracutaneously to another person a local reaction of delayed type is seen at the site of transfer 24-48 hr later (‘normal lymphocyte transfer reaction’). When sensitized lymphocytes of the recipient afterwards are transferred back to the donor in normal cases the local reaction is much stronger (‘immune lymphocyte transfer reaction’). When the recipients were patients with acute sarcoidosis or with Hodgkin’s disease of stage III and IV, the normal lymphocyte transfer reaction was considerably weaker than in normal cases. The reaction of patients with chronic sarcoidosis was similar to normal controls, likewise the reaction of early cases of Hodgkin’s disease. In back transfer of sensitized lymphocytes of above mentioned recipients to the donors the immune lymphocyte transfer reaction was stronger in acute sarcoidosis than in stage III of Hodgkin’s disease, but always weaker than in normal cases. The reactions of patients with chronic sarcoidosis were similar to normal controls. The investigations show a parallel to the findings with passive transfer of delayed type sensitivity.

Lymphocyte-induced angiogenesis: a quantitative and sensitive assay of the graft-vs.-host reaction.

A new and sensitive assay for the effect of intracutanous administration of immunocompentent lymphocytes into the skin of irradiated unimmunized mice is described. The assay, which we have termed lymphocyte-induced angiogenesis (LIA) involves enumeration of new vascular branches induced by the action of these competent cells. As is the case for the previously described normal lymphocyte transfer reaction, LIA is a manifestation of the graft-vs.-host reaction, as shown by experiments utilizing appropaiate genetic combinations. The reaction is dose-dependent, and within the dose range of 2 times 10 minus 5 -4 times 10-6 cells the mumber of vessels induced correlates with the mumber of immunocompetent cells injected. At these dose levels spleen, lumph node, and hydrocortisone-resistant thymocytes are effective; bone marrow and thymus cells are not. Spleen cells from nude mice are incapable of inducing LIA, while mitomycin-C and irradiated lymphocytes can initiate but not maintain the reaction. The relationship between lymphocyte-induced angiogenesis has been discussed as have the implications of these findings to delayed hypersensitivity, inflammation, and vascular pathology.

Vitamin B 6 deficiency and the lymphoid system : I. Effects on cellular immunity and in vitro incorporation of 3H-uridine by small lymphocytes

Thoracic duct lymphocytes from vitamin B 6-deficient rats were found to have a reduced capacity to respond to foreign lymphoid cells in the mixed lymphocyte reaction (MLR), to produce normal lymphocyte transfer reactions, and to incorporate 3H-uridine in vitro. These findings indicate that specific nutritional deficiencies may impair cellular immunity and that this impairment can be monitored by the MLR. It is suggested that the reduction in MLR activity and in 3H-uridine uptake by TDL cells reflected either a shift in the proportions of T and B cells in the TDL and/or an impairment in the capacity of such cells to function in the MLR and in the in vitro test for 3H-uridine incorporation.

Vitamin B 6 deficiency and the lymphoid system : II. Effects of vitamin B 6 deficiency in utero on the immunological competence of the offspring

Results of this study indicated that an absence of vitamin B 6 from the diet of pregnant rats led to reduced immunological competence in the offspring. While the numbers of cells in the thoracic duct lymph (TDL) of rats approximately 3 months old and progeny of vitamin B 6-deficient mothers were nearly equivalent to control values, such cells had a reduced capacity to respond in the mixed lymphocyte and normal lymphocyte transfer reactions. It is suggested that this reduction may have reflected (i) an alteration in the capacity of TDL cells or their precursors to give rise to immunologically competent cells, (ii) a shift in the proportions of T and B cells in the TDL, and/or (iii) an absence or ineffectiveness of a humoral factor required for the development of immunologically competent lymphocytes.

Differences in the graft-versus-host reactivity of cells migrating through nonlymphoid tissue or lymph nodes.

Normal lymphocyte transfer reactions have been used to test the alloreactivity of lymphocytes which have migrated through lymph nodes (i.e., in efferent lymph) or nonlymphoid tissues (i.e., in afferent lymph) in sheep. Lymphocytes from afferent lymph are much less efficient at inducing a normal lymphocyte transfer lesion than lymphocytes from efferent lymph. Afferent cell preparations (which contain about 10% macrophages) do, however, cause a greater inflammatory response in the early part of the reaction, but this is at least partly nonspecific. A mixture of afferent and efferent cells produces a lesion which is simply the sum of the two expected reactions, suggesting that the afferent lymphocytes are not merely inhibited by a factor or cell in the preparation.

An in vivo test for the immunocompetence of human lymphocytes.

After the injection of mice with human lymphocytes suspended i.d. in Medium 199 a local area of induration was obtained. This was maximal at 48 hr and correlated to the number of lymphocytes injected. Evidence that this normal lymphocyte transfer reaction was graft-versus-host in character was the finding that the reaction size was independent of the administration of rabbit antimouse thymocyte serum to the recipient mice. However, the reaction could be suppressed by administration of rabbit antihuman lymphocyte serum. Lymphocytes from 7 of 10 patients with advanced carcinoma of the urinary bladder, 4 patients with advanced carcinoma of the breast, and 1 case each of carcinoma of the prostate, carcinoma of the uterus, and malignant melanoma showed diminished normal lymphocyte transfer reactivity. This was assessed both in relation to patients of similar age with debilitating nonneoplastic conditions and to patients in a surgical ward whose general condition was good.

The mechanism of action of antilymphocytic serum: an electron microscopic study

Antilymphocytic serum (ALS) was prepared from rabbits immunized against Lewis rat lymph node and thymus gland cell suspensions. The antiserum was shown to be immunosuppressively active by the normal lymphocyte transfer reaction. Intraperitoneal injection of active antilymphocyte serum into male Lewis rats resulted in the following findings. First, peripheral blood lymphocyte counts were depressed up to 95%. Second, extensive phagocytosis of lymphocytes was observed from 2–4 hr after treatment. This phagocytosis was more prominent in the liver followed by lymph node and spleen. Third, the phagocytic process included engulfment of the lymphocytes, lysosomal association with the phagosomes, presumably release of enzymes into the phagosomes, and lymphocyte degradation. Fourth, erythrocytes were also phagocytized. The implications of these findings were discussed in relationship to the proposed mechanisms of action of the ALS.