CD4−8− T-cells increase in MRL/lpr mice treated with thymic factors

The in vivo effect of thymic factors on immature lymphocytes was analysed in MRL/lpr mice. This strain carries a genetic defect that causes during their life cycle a block of T-cell differentiation and abnormal proliferation of CD4⁻8⁻ (double-negative, DN) T-lymphocytes. In vivo administration of four preparations of thymic factors, thymopentin (TP-1), thymopoietin (TP-5), thymolymphotropin (TLT), and thymomodulin (TMD) into young (2-month-old) MRL/lpr mice induced a significant increase of DN T-cells both in the thymus and in the peripheral lymph nodes, with a concomitant decrease of double-positive (DP) T-cells in the thymus and of single-positive (SP) T-cells in the lymph nodes. The level of DNA fragmentation measured as propidium iodide fluorescence was increased in the thymus population of young mice and in the lymph node population of old mice treated with TLT. SCID mice transplanted with lymph node cells from MRL/lpr donors (MRL→SCID) developed graft versus host (GvH) reaction due to the activation of MRL CD8⁺ alloreactive T-cells. This model was used to analyse the effect of TMD/TLT in vivo on MRL cell proliferation and expansion; in fact, spleen cells from MRL→SCID mice after treatment with TMD/TLT showed an increased cell proliferation, and an expansion of DN T-cells with a concomitant decrease of SP cells (both CD4⁺ and CD8⁺ cells). Decreased SP cell numbers in this context could explain why TMD/TLT treatment of SCID mice engrafted with MRL cells increased their survival compared to untreated MRL→SCID mice.     

Enhancement of various non-specific immune effector functions in mice by local injection of aclacinomycin A adsorbed onto activated carbon particles (ACR-CH)

Local injections of aclacinomycin A adsorbed onto activated carbon particles (ACR-CH) augmented the cytotoxic activities of regional lymph node cells for 7 days. In contrast NK-activity was only slightly augmented by injections of aclacinomycin A (ACR) solution or activated carbon suspension. The effects were found in lymphocytes from all regions tested. NK-activity could only be detected when both adherent and non-adherent cells were present. The cell number of L3T4⁺ cells in each type of lymph node tested increased, and subset analysis of the lymphocyte subpopulations revealed an increase in the ratio of L3T4⁺/Lyt2⁺ cells, suggesting that the ACR-CH selectively increased and stimulated L3T4⁺ cells. Enhanced capacity of lymph node cells to produce cytokines, tumor necrosis factor (TNF) and interleukin-1 (IL-1) upon restimulation (with LPS) in vitro in the ACR-CH treated group was found. From these results, it appears that the new dosage form of aclacinomycin A, ACR-CH, with superior therapeutic efficacy against lymph node metastases, can also enhance the immune response of regional lymph node cells. The findings reported here will be valuable in the establishment of novel chemoimmunotherapeutic protocols using ACR-CH.     

The cytotoxic activity of lymphocytes from human lymph in vitro

The in vitro cytotoxicity and DNA synthesis of thoracic duct and blood lymphocytes from four patients have been studied on the 1st day of drainage. Three patients were being drained as a pretreatment for kidney transplantation and one had myasthenia gravis. In one patient lymphocytes were obtained from a lymphatic fistula in the groin and from the blood 5 weeks after drainage began. Lysis of tissue culture cells (Chang cells) in the presence of PHA or antiserum to target cell antigens was quantitated by [⁵¹Cr]chromate release.

Lymphocytes from lymph were at best poorly cytotoxic to antibody-treated target cells under conditions where purified blood lymphocytes from the same donors had normal lytic activity. PHA-induced cytotoxicity by lymph-borne lymphocytes was noted but was considerably weaker than that of blood lymphocytes. In contrast, incorporation of [¹⁴C]thymidine into DNA of thoracic duct lymphocytes after stimulation with PHA was about 60% of that of the patients' blood lymphocytes. The DNA synthesis of thoracic duct lymphocytes induced by PPD or allogeneic lymphocytes was as good as that of blood lymphocytes. The mitotic response to PHA by lymphocytes from the lymph was reduced after two weeks drainage.

It is assumed that the number of effector cells and/or supporting cells in antibody-induced cytotoxicity in thoracic duct lymph is too small to induce target cell lysis under the present experimental conditions. Moreover, our data indicate that PHA-induced and antibody-mediated cytotoxicity are at least partly mediated by different lymphocyte subpopulations.

     

Lymphopoiesis and lymph node histogenesis in the embryonic and neonatal rabbit

The thymus represents the first lymphocytic organ to exhibit lymphopoietic activity in the fetal rabbit with lymphoblastic transformation beginning by 17 days of gestation, and lymphocytes appearing by 18 days, one to two days before vascularization. Lymphocytes are first evident in lymph nodes at 18 days of fetal development, in the spleen on the twenty-third day, and in the appendix just prior to birth. Medium-sized lymphocytes and rare blast-like cells represent the first lymphocytic cells found in the lymph node anlage. Both of these cell forms appear simultaneously and are distributed randomly. Lymphocytes remain relatively sparse increasing gradually in number until several days prior to birth when their numbers increase rapidly. Coincident with the augmentation in lymphocyte population, lymphocytes tend to cluster near the vascular channels, and are present within the lumina and walls of the smaller veins in the node. Morphological gradations between mesenchymal cells and medium-sized lymphocytes provide suggestive evidence that the initial population of lymphocytes within the node arise by the direct transformation of the stromal cells into medium-sized lymphocytes. Less frequently, deeply basophilic blast-like cells may provide an intermediate stage in lymphocyte differentiation. These cells differ from typical lymphoblasts because of their smaller size and irregular cellular contour. It is postulated that the initial population of lymphocytes formed in the lymph node anlage is derived from the transformation of mesenchymal cells and subsequent homoplastic lymphocyte proliferation and that during later fetal life this population of lymphocytes is complemented by the colonization and proliferation of blood-borne lymphocytes.     

Lymphatic mast cell response and effect of compound 48/80 on popliteal lymph node reaction in rats following intracutaneous injection ofStaphylococcus aureus

To investigate the significance of mast cells in the popliteal lymph node during the development of an inflammatory response, rats were inoculated with 12×10⁷ colony-forming units ofStaphylococcus aureus in the hind foot pad. Numerical changes in mast cells were then measured in the corresponding popliteal lymph node. Six days after inoculation, despite the enlargement of the responding lymph node, a marked decrease in granulated mast cell number, relative to the contralateral node, was observed in the cortical and medullary compartments. Popliteal lymph nodes from rats treated with compound 48/80 and then inoculated withS. aureus showed a higher cortical and medullary hypertrophic response and a significant increase in degranulated/weakly basophilic mast cell number in the lymph node tissue. The findings suggest that (1)Staphylococcus aureus induces a reduction in granulated mast cell number in the cortical and medullary compartments of regional lymph nodes; (2) pretreatment with compound 48/80 appears to contribute to the lymphoid cell proliferation and the hypertrophic response of lymph nodes induced byS. aureus; and (3) granulated mast cells have a regulatory role on lymphoid cell proliferation.     

The role of non-specific lymphocytes in antigen-induced proliferative response in vitro

The antigen-induced proliferative response of lymph node cells from immunized mice is proportional to the number of primed lymphocytes in the microtiter wells. Addition of normal lymphocytes did not alter the magnitude of the antigen-specific [³H]TdR uptake of immune lymph node cells. In contrast, normal lymphocytes increased the antigen-specific thymidine incorporation of enriched populations of antigen-specific lymphocytes. This enhancing effect was especially pronounced, and proportional to the number of supplementing unsensitized lymphocytes, with a small cell number of enriched lymphocytes, where the specific responsiveness could barely be detected without addition of normal lymphocytes.Enriched populations derived through adherence to antigen-pulsed macrophages (‘selected’ cultures) as well as those obtained by growing immune lymph node cells with antigen-pulsed macrophages (‘supernatant’ cultures) had an improved proliferative response after addition of normal lymphocytes. However, the proliferative response of the ‘selected’ cultures was extremely dependent on normal lymphocytes as they could express only 10% of their full stimulatory capability in the absence of the latter. This indicates that the blastogenic signal delivered by the activated specific T cells recruits normal lymphocytes which do not adhere to the antigen-pulsed macrophages. Under the same conditions the recruiting signal is incapable of inducing the multiplication of antigen-sensitized cells.The potential recruitable uncommitted lymphocytes are present in excess among immune lymph node cells, while depleted from the ‘selected’ cultures. The enriched ‘supernatant’ cultures seem to contain considerable numbers of recruitable lymphocytes although they are present in quantities less than are required to provide for the full amplifying signal.This meaning of these findings for the evaluation of enrichment of antigen-specific T cells and of antigen-induced response based on [³H]TdR uptake is discussed.     

Proliferative response to the skin-sensitizing contact agent dinitrofluorobenzene in lymph nodes in mice

When dinitrofluorobenzene (DNFB) is applied to the skin in mice, it induces a state of delayed hypersensitivity. During the three days following the application of this contact agent, lymphoblasts accumulate in the diffuse cortex of the lymph node draining the area of sensitization. This accumulation of blast cells – referred to as a blastogenic response – appears to be part of the induction phase of delayed hypersensitivity. This is an investigation into the origin of the cells of the blastogenic response. There is evidence from other studies that lymphocytes immigrate to the lymph nodes draining a site of sensitization. This paper provides complementary evidence that cellular proliferation in the diffuse cortex of the sensitized lymph node is another source for the accumulation of blast cells. This proliferation has been studied by counting mitotic figures, and by autoradiography after giving ³H-thymidine in order to determine the number of blast cells undergoing DNA synthesis and the duration of the various phases of the cellular reproductive cycle. The results show a high degree of correlation between mitotic figures and blast cells in the diffuse cortex of the sensitized lymph nodes, consistent with a population of dividing blast cells. Approximately 80 percent of the blast cells were in the DNA-synthetic phase of the reproductive cycle at the peak of the blastogenic response, and the cells were dividing with approximately an eight hour generation time. These results can be interpreted to indicate that the cells of the blastogenic response, although accumulating initially by immigration to the draining lymph node, become a homogeneous, nondifferentiating population of blast cells, all proliferating at near maximal rate to provide the great numbers of blast cells seen three days after application of the sensitizing agent.     

Control mechanism of lymphocyte traffic Altered migration of 51Cr-labeled mouse lymph node cells pretreated in vitro with phospholipases

Glycoproteins have so far been the only surface components thought to be of importance in the process of recognition between circulating lymphocytes and endothelial cells of the lymph node post-capillary venules and thus in the control of lymphocyte traffic. In this paper the effect of in vitro treatment of ⁵¹Cr-labeled mouse lymph node cells with phospholipases (PL) (A and C) on their migration into syngeneic recipients was investigated. PL-A and PL-C-treated cells migrated differently from control (untreated) cells. Diminished numbers of PL-A-treated lymphocytes were found in the lymph nodes at 1, 6 and 24 h after cell transfer with a simultaneous increase in the lungs at 1 h, and spleen at 6 h after transfer. PL-C-treated cells remained in the blood longer than untreated cells. We conclude that factors other than the integrity of surface glycoproteins are involved in the control of lymphocyte traffic. The roles played by cell adhesion, in particular cell-to-cell interactions in regulating the rate of cell migration, are discussed.     

Migration of newly formed small lymphocytes from bone marrow to lymph nodes during primary immune response

Selective DNA labelling of bone marrow cells in vivo was used to determine the effect of antigenic stimulation on the migration of small lymphocytes from bone marrow to popliteal lymph nodes. Following footpad injection of keyhole limpet haemocyanin (KLH) in guinea-pigs the regional nodes showed an early increase in weight and cellularity together with a progressive increase in cell proliferation. When [³H]thymidine was injected into tibial and femoral marrow 2 days before KLH administration the DNA radioactivity of the KLH-stimulated nodes increased rapidly and always exceeded that of contralateral nodes. Simultaneously, in radioautographic sections of lymph nodes labelled small lymphocytes, indicative of an origin from marrow precursors, appeared throughout the cortex, post-capillary venules, subcapsular sinus, medullary cords and sinuses. In KLH-stimulated nodes the number of labelled small lymphocytes per section was higher than in contralateral nodes, especially in the cortex, and some of these cells appeared in germinal centres. Labelled large blast cells and macrophages were also increased in numbers. Similar changes were observed in lymph nodes of parental strain rats following intramyeloid [³H]thymidine administration and footpad injection of lymphoid cells from F1 hybrid rats. The results demonstrate that, during the early response of lymph nodes to various antigens, local changes in cell traffic include an enhanced accumulation of newly formed small lymphocytes, putative virgin B lymphocytes, generated in the bone marrow prior to the antigenic stimulation.     

Phenotype, functions and fate of adoptively transferred tumor draining lymphocytes activated ex vivo in mice with an aggressive weakly immunogenic mammary carcinoma

Background

Regression of established tumors can be induced by adoptive immunotherapy with tumor draining lymph node lymphocytes activated with bryostatin and ionomycin. We hypothesized that tumor regression is mediated by a subset of the transferred T lymphocytes, which selectively infiltrate the tumor draining lymph nodes and proliferate in vivo.

Results

Adoptive transfer of B/I activated tumor draining lymphocytes induces regression of advanced 4T1 tumors, and depletion of CD8, but not CD4 T cells, abrogated tumor regression in mice. The predominant mediators of tumor regression are CD8+ and derived from CD62L^- T cells. Transferred lymphocytes reached their peak concentration (10.5%) in the spleen 3 days after adoptive transfer and then rapidly declined. Adoptively transferred cells preferentially migrated to and/or proliferated in the tumor draining lymph nodes, peaking at day 5 (10.3%) and remained up to day 28. CFSE-stained cells were seen in tumors, also peaking at day 5 (2.1%). Bryostatin and ionomycin-activated cells proliferated vigorously in vivo, with 10 generations evident in the tumor draining lymph nodes on day 3. CFSE-stained cells found in the tumor draining lymph nodes on day 3 were 30% CD8⁺, 72% CD4⁺, 95% CD44⁺, and 39% CD69⁺. Pre-treatment of recipient mice with cyclophosphamide dramatically increased the number of interferon-gamma producing cells.

Conclusions

Adoptively transferred CD8+ CD62L^low T cells are the principal mediators of tumor regression, and host T cells are not required. These cells infiltrate 4T1 tumors, track preferentially to tumor draining lymph nodes, have an activated phenotype, and proliferate in vivo. Cyclophosphamide pre-treatment augments the anti-tumor effect by increasing the proliferation of interferon-gamma producing cells in the adoptive host.     

Differential Regulation of Mouse B Cell Development by Transforming Growth Factor β1

Transforming growth factor β (TGFβ) can inhibit the in vitro proliferation, survival and differentiation of B cell progenitors, mature B lymphocytes and plasma cells. Here we demonstrate unexpected, age-dependent reductions in the bone marrow (BM) B cell progenitors and immature B cells in TGFβ1^-/- mice. To evaluate TGFβ responsiveness during normal B lineage development, cells were cultured in interleukin 7 (IL7)±TGFβ. Picomolar doses of TGFβ1 reduced pro-B cell recoveries at every timepoint. By contrast, the pre-B cells were initially reduced in number, but subsequently increased compared to IL7 alone, resulting in a 4-fold increase in the growth rate for the pre-B cell population. Analysis of purified BM sub-populations indicated that pro-B cells and the earliest BP1^- pre-B cells were sensitive to the inhibitory effects of TGFβ1. However, the large BP1⁺ pre-B cells, although initially reduced, were increased in number at days 5 and 7 of culture. These results indicate that TGFβ1 is important for normal B cell development in vivo, and that B cell progenitors are differentially affected by the cytokine according to their stage of differentiation.     

Effects of heterologous anti-lymphocyte serum on the distribution of 51Cr-labelled lymph node cells in mice

The effects of heterologous anti-lymphocyte serum (ALS) were studied in a syngeneic cell transfer system, in which lymph node cells from donor CBA mice were labelled in vitro with ⁵¹Cr and transferred intravenously into syngeneic recipients. Labelled cells treated in vitro with ALS were unable to migrate to lymph nodes or spleens of recipients, as did normal cells, but instead distributed themselves very similarly to cells which had been killed by exposure to heat. It is thus likely that cells treated in vitro with ALS are killed after transfer by the cytotoxic action of ALS mediated by the complement of the recipient.

ALS administered directly to the recipients of labelled lymphocytes could also reduce their uptake into lymphoid tissue; however, the magnitude of this effect appeared to be critically dependent upon the timing of the antiserum dose with respect to the labelled cell dose. ALS given immediately prior to labelled cells showed the greatest effect, while treatment given either 24 hours before or after the labelled cells was much less effective. While with prior treatment the reduced effect could be due to a fall off in antibody titre during the interval between the dose of antiserum and cells, in the latter situation no drop in titre would have occurred. It thus seems that lymphocytes that have already established themselves in lymphoid tissue may be less susceptible to the action of ALS.

ALS given chronically to lymphoid cell donors resulted in a population of cells which upon transfer to normal recipients were distributed differently from either normal or NRS-treated donor cells.

These data support the hypothesis that the effects of ALS may be exerted preferentially on circulating lymphocytes, and that ALS may act to selectively reduce the representation of this cell type in lymphoid tissue.

     

Quantitative Studies on the Proliferation and Differentiation of Antibody-Forming Cells in Lymph

The transforming cells that appear in the efferent lymph from a lymph node responding to an antigenic challenge are part of a heterogeneous population which changes as the response progresses. Some cells containing small amounts of antibody appear early in the response and these cells have the cytologic characteristics of small and medium lymphocytes. They are, however, actively synthesizing DNA. As the immune response progresses, the antibody content of the cells in lymph increases. When incubated in vitro, cells in lymph appearing late in the response released 20 times more antibody per cell than those appearing early in the response. Large blast cells are the predominant antibody-forming cell in lymph. At the peak of a secondary challenge with horseradish peroxidase, up to 40% of the cells in lymph may be blast cells and, of these, two-thirds may contain specific antibody. It seems probable that most if not all of the blast cells responding to the antigen are involved directly in antibody and DNA synthesis. Cells in all stages of ultrastructural differentiation, and even mature plasma cells, were found to incorporate ³H-thymidine into their nuclear DNA.     

Fate of Autogenous Canine Lymphocytes after Injection into an Afferent Lymphatic of the Popliteal Lymph Node

Dog thoracic duct lymphocytes were labeled in vitro with ³H-uridine and infused into an afferent lymphatic of the popliteal lymph node of the same dog. Ten minutes after infusion nearly all the injected radioactivity was recovered from the lymph node. An effect of infusion flow rate on the percentage of cells retained by the lymph node was observed ½ to 3½ hours after infusion, and was probably mediated by the tendency of the node to become edematous after infusions at a rate exceeding 0.045 ml/min. Edematous nodes retained 83.7% of the cells, as compared to 47.5% for nonedematous nodes. As early as 30 minutes after infusion a small amount of ³H-radioactivity was found in the spleen and thoracic duct lymph. The deep iliac and paraaortic nodes on the side of the infusion contained significant amounts of ³H-radioactivity, while negligible amounts were detected in the contralateral popliteal node at any time. The intranodal localization of the ³H-labeled cells was studied by radioautography. All labeled cells remained intrasinusoidal during the first 4 hours after infusion. At 9 and 21 hours some labeled cells were located in the extrasinusoidal parenchymal lymphoid tissue of the cortex and the medulla, but the majority still remained intrasinusoidal.     

The effect of localized injection of adjuvant material on the draining lymph node: II. Circulating lymphocytes

The stimulation of a draining lymph node by the injection of a substance possessing adjuvanticity leads to a marked increase in the influx of lymphocytes into the node. The influx of cells has been followed in experimental mice injected intravenously with ⁵¹Cr-labelled normal mesenteric lymph node cells. The increase in size of the draining node is not due to an increased blood volume.     

Difference between the lymph node response to injection of autosensitized T lymphocytes and that in a graft-versus-host reaction

Thymus (T) lymphocytes autosensitized in vitro were shown in previous studies to produce enlargement of draining popliteal lymph nodes upon injection into the footpads of syngeneic rats. Specific autoreactive effector lymphocytes were found to be recruited within these lymph nodes. In the present study, the cellular basis of lymph node enlargement in mice by autosensitized lymphocytes was compared with that produced in a graft-vs.-host (GvH) reaction. T lymphocytes of C3H mice were autosensitized against syngeneic fibroblasts in vitro for 16 to 18 h in the absence of serum, and 10⁷ lymphocytes were injected into the footpads of syngeneic mice. Control lymphocytes were incubated without fibroblasts. The GvH reaction was produced by injecting 10⁷ C3H T lymphocytes into the footpads of (C3H × C57BL)F₁ adult recipients. The index of relative enlargement of the draining popliteal lymph nodes was measured 6 days after injection. Experiments were done to identify the origin of the lymph node cells in these reactions. Irradiation of the donor lymphocytes (1000 r) or the recipient mice (550 r) was used to prevent proliferation of the lymphocytes of either origin. The participation of recipient T lymphocytes in lymph node enlargement was investigated by using thymectomized mice. The following results were obtained. 1) The GvH lymph node enlargement was found to depend on proliferation of the donor T lymphocytes, but did not seem to require the participation of radiosensitive cells within the recipient mice. 2) In contrast, the response of the lymph nodes to autosensitized donor T lymphocytes depended on the function of radiosensitive T lymphocytes within the syngeneic recipients. The autosensitized donor lymphocytes themselves did not have to proliferate to recruit the response of recipient T lymphocytes. 3) It was found that recruitment of recipient lymph node cells could be super-imposed upon a conventional GvH reaction by presensitizing the C3H donor lymphocytes in vitro. Both autosensitization against syngeneic or allosensitization against C57BL fibroblasts augmented the lymph node response of (C3H × C57BL)F₁ hybrid recipients. The recruited or donor components of these mixed responses could be selectively abolished by irradiating either the donor lymphocytes or the recipient mice. Hence, the autosensitization response, like the host-vs.-graft transplantation reaction, can be induced by sensitization of lymphocytes peripherally and involves recruitment of lymphocytes within regional lymph nodes. The GvH response manifested in the same popliteal lymph nodes does not appear to require the recruitment of radiosensitive T lymphocytes. These findings suggest that different classes of T lymphocytes function in the autosensitization and GvH responses.     

Mast cell responses in mesenteric lymph nodes to infection of rats with the nematode, Nippostrongylus brasiliensis

The number of mast cells and their distribution in rat mesentery lymph nodes were assessed after a primary infection and after several successive infections with the nematode, Nippostrongylus brasiliensis. Following primary infection with N. brasiliensis, two peaks in total mast cell counts were observed. An initial small increase was restricted to day 5 and to the region of entrance to the lymph node. During the second peak, a marked increase in the number of mast cells occurred after day 15, the majority of cells is migrating through the afferent lymphatics, and then advancing from the cortical to the medullary region. The number of cells found in the hilus always remained low, indicating that mast cells accumulate and degranulate within the lymphoid organ.

In rats infected several times with the nematode parasite, mast cell numbers were markedly increased and the distribution pattern was similar to that found on day 21 after a primary infection. The observation that the percentage of cells found in the capsule was rather low in these animals indicates that local proliferation might have contributed to the high mast cell counts.

     

Delayed hypersensitivity to Staphylococcus aureus in mice: kinetics of induction

The induction of systemic delayed hypersensitivity to Staphylococcus aureus is dependent on a minimum of two weekly injections of viable bacteria. In vitro lymphocyte stimulation studies show that the spleen acts as a repository for the immunogen-responsive lymphocytes associated with DH. To evaluate the kinetics of induction, mice were given one to three weekly injections of viable S. aureus and the weights, lymphocyte numbers, DNA synthesis and lymphocyte immunogen reactivity of the draining lymph node (DLN), contralateral lymph node (CLN), and spleen (SP) were determined at days 7, 14 and 21 post injection. The staphylococcal immunogens included whole cell sonicate, cell wall, cell membrane, protein A, lipoteichoic acid, teichoic acid and purified membrane proteins. A single injection resulted in an increase in organ weight, lymphocyte numbers and DNA synthesis of the DLN. This was accompanied by lymphocyte responsiveness to all immunogens. There was an increase in spleen weight and lymphocyte numbers without an appreciable increase in DNA synthesis. Spleen lymphocytes showed no increase in immunogen responsiveness. A second injection maintained the increased weight, lymphocyte numbers and DNA synthesis of the DLN but resulted in a suppression of lymphocyte responsiveness to all immunogens. Splenic lymphocytes showed in vitro reactivity to all immunogens. This was accompanied by an increase in lymphocytes and mitogenic responsiveness without in vivo mitosis. The third injection maintained suppression of DLN cells and further stimulated those of the spleen. The splenic lymphocytes were hyper-reactive to B-lymphocyte and T-lymphocyte mitogens and showed an increased rate of DNA synthesis.     

Age-related changes in localization of injected radiolabelled lymphocytes in the lymph nodes of antigen-stimulated mice.

The localization of i.v. injected syngeneic lymph node cells, radiolabelled with 51Cr or 75Se-L-selenomethionine, was studied in male CBA/H mice aged between 3 and 30 months. The following results were obtained. (1) Localization of cells from young adult donors was greater in the s.c. lymph nodes of old than of young recipients, the main increase being between 15 and 17 months of age. Increases in lymph node weight and DNA-synthesis were also seen at this time; but the rise in cell localization was significant even when calculated per unit of tissue weight. Splenic localization either declined slightly with age or, like the liver, showed no significant change. (2) Local antigenic stimulation by a single injection of sheep erythrocytes into one front footpad, 24 hr before lymph node cell injection, resulted in increased localization in the regional lymph nodes of 3-17 month old, but rarely of 24-30 month old mice. (3) No consistent differences in localization were observed between lymph node cells from 4-month and 25-month old donors. Both age-related and antigen-related increases in cell localization were at least partly attributable to an enhanced rate of entry of lymphocytes from the blood to the lymph nodes. Although the changes underlying the decline in antigen-related localization of cells in old recipients have still to be clarified, it is probable that the defective immune responses of old mice result partly from this decline.     

T lymphocyte proliferation is suppressed by the opioid growth factor ([Met(5)]-enkephalin)-opioid growth factor receptor axis: implication for the treatment of autoimmune diseases

Opioid peptides function as immunomodulatory molecules. Reports have linked the opioid growth factor (OGF), [Met⁵]-enkephalin, and its receptor OGFr to autoimmune diseases. OGF repressed the incidence and magnitude of myelin oligodendrocyte-induced experimental autoimmune encephalomyelitis in mice. Given the extensive connection between the immune system and autoimmune diseases, the present study was conducted to examine the relationship of the OGF-OGFr axis and T lymphocyte proliferation. Splenic-derived mouse lymphocytes were stimulated with phytohemagglutin (PHA). All non-stimulated and PHA-stimulated T lymphocytes had immunoreactivity for OGF-like enkephalin and OGFr. OGF markedly suppressed T lymphocyte number in a dose-dependent manner. However, PHA-stimulated T lymphocytes were not altered in cell number by a variety of natural and synthetic opioid-related compounds, some specific for μ, δ, and κ opioid receptors. Persistent blockade of opioid receptors with the general opioid antagonist naltrexone (NTX), as well as antibody neutralization of OGF-like peptides, had no effect on cell number. Non-stimulated T lymphocytes exhibited no change in cell number when subjected to OGF or NTX. Treatment of T lymphocytes with siRNAs for μ, δ, or κ opioid receptors did not affect cell number, and the addition of OGF to these siRNA-exposed cultures depressed the population of cells. T lymphocytes treated with OGFr siRNA also had a comparable number of cells to control cultures, but the addition of OGF did not alter cell number. DNA synthesis in PHA-stimulated T lymphocytes exposed to OGF was markedly decreased from PHA-stimulated cultures receiving vehicle, but the number of cells undergoing apoptosis or necrosis in these cultures was similar to control levels. T lymphocytes subjected to siRNA for p16 and/or p21 had a comparable number of cells compared to controls, and treatment with OGF did not depress cell number in preparations transfected with both p16 and p21 siRNA. These data reveal that the OGF-OGFr axis is present in T lymphocytes and is capable of suppressing cell proliferation. However, T lymphocytes are not dependent on the regulation of cell proliferation by this system. The results showing that the OGF-OGFr axis is an immunosuppressant, offers explanation for reports that autoimmune diseases can be modulated by this system.