Antibody-dependent cellular cytotoxicity in the guinea pig: The role of the Kurloff cell

In the guinea pig, the killer cell in in vitro ADCC assays, is found to be the Fc receptor-bearing Kurloff cell. This killer Kurloff cell is under hormonal control, estrogen treatment significantly increasing the Kurloff cell numbers in blood, spleen, and thymus, and markedly augmenting the lytic capacity of these lymphoid compartments. The cytotoxic killer lymphocyte, if present, appears to be a minor effector cell in guinea pig ADCC. Selective lymphocyte subpopulation and Kurloff-cell depletion procedures reveal that the killer Kurloff cell may also possess a variety of other membrane markers (T⁺, C3⁺, Ig⁺). The particular membrane profile of a cytotoxic Kurloff cell is determined by its lymphoid site of residence and the hormonal status of the animal.     

Characterization of HNK-1+ (Leu-7) human lymphocytes: III. Interferon effects on spontaneous cytotoxicity and phenotypic expression of lymphocyte subpopulations delineated by the monoclonal HNK-1 antibody

Interferon (IFN) augments the cytotoxic function of natural killer (NK) and killer (K) cells. We have previously shown that all NK- and K-cell function resides in the HNK-1⁺ population of granular lymphocytes. The present experiments demonstrated that IFN significantly augmented the efficiency of purified HNK-1⁺ cells to perform both NK- and K-cell function. In contrast, HNK-1^? cells could not lyse target cells even in the presence of IFN. IFN did not generate new HNK-1⁺ cells from the pool of HNK-1^? cells. We then examined the possibility that IFN might induce the maturation of immature NK cells previously defined as having an HNK⁺T3⁺ phenotype and a paucity of cytoplasmic granules. However, no changes were observed either in the proportion of HNK-1⁺ cells expressing the T3 antigen or in the number of granules within each HNK-1⁺ cell even after an 18-hr incubation with IFN. While fresh HNK-1^? cells lack NK-cell function, they can acquire NK-like activity without expressing the HNK-1 antigen after incubation with either alloantigens or mitogens. When incubated further with IFN, these alloantigen- or PHA-activated HNK-1^? cells with NK-like activity demonstrated relatively little augmentation of their cytotoxicity. It is concluded that interferon exerts its influence on restricted subpopulations of cells, primarily HNK-1⁺ cells. Its mechanism appears to concern the cytotoxic event rather than influencing cellular maturation.     

Subpopulations of human peripheral blood lymphocytes.

Multiple staining protocols have been developed for the classification of subpopulations of human peripheral blood lymphocytes. Of the non-T (E^?) cells, roughly half (10–20% PBL) have receptors for complement components as detected with complement-coated zymosan particles, but do not show Fc receptors as detected with Ripley IgG-coated human RBC. The other half are C^?, Fc⁺, with a small percentage possessing both receptors. The C⁺, Fc^? cells can be subdivided into cells which are IgM⁺ (75%) or IgM^?. Cells with Fc receptors detected with aggregated IgG were IgM⁺.     

Surface phenotype of responder cells in the syngeneic mixed lymphocyte reaction in mice

The organ distribution and surface phenotype of SMLR responder cells has been investigated. Nylon-wool-passed spleen cells, which proliferate in response to mitomycin-C-treated syngeneic spleen cells, are Thy 1.2⁺ Ly 1⁺2^?3^?. SMLR responder cells are not confined to the spleen since nylon-wool-nonadherent lymph node cells as well as unfractionated thoracic duct lymphocytes show activity. Responder cells have characteristics of mature T cells since cortisone-resistant thymocytes, but not thymocytes from untreated mice, are capable of SMLR response. In addition, naturally occurring thymocytotoxic antibody (NTA), which in our experiments exhibits cytotoxicity only for thymocytes, does not appear to affect the subpopulation of the T cells which respond in the SMLR.     

Lysis of herpes simplex virus-infected targets: II. Nature of the effector cells

Peripheral blood lymphocytes (PBL) from patients with herpes simplex virus (HSV)-1 recurrences in the cornea only (Group I) exhibited reduced lysis of HSV-1-infected targets compared to PBL from patients with oral-facial and corneal HSV recurrences (Group II). The cytotoxic lymphocytes appeared to belong to a subpopulation of natural killer (NK-HSV) cells. Monoclonal antibodies to human lymphocyte differentiation antigens were used to define the surface phenotype of the NK-HSV cells. Most of the NK-HSV activity was mediated by lymphocytes expressing the surface markers Leu-7⁺ (HNK-1), OKT3⁺ (pan T), OKM1⁺ (myeloid and NK), Leu-2^? (cytotoxic/ suppressor T cell), and Leu-8^? (regulatory T cell). In contrast, lysis of K562 cells (NK-K562) was mediated by lymphocytes bearing the surface phenotype Leu-7⁺, OKT3^?, OKM1⁺, Leu-2^+/?, and Leu-8^?. The low level of NK-HSV activity in PBL from Group I donors appeared to be due to active suppression by suppressor T lymphocytes. Depletion of Leu-2⁺ cells from PBL of Group I donors resulted in significant augmentation of NK-HSV activity. Similar treatment of PBL from Group II donors either had no effect or slightly diminished the NK-HSV activity.     

Immunological properties of Fc receptor on lymphocytes. 2. Differentiation from FcR- to FcR+ cells and their functional differences in in vitro antibody response.

In in vitro plaque-forming cell (PFC) response to particulate as well as to soluble antigen, the functional difference between Fc receptor-bearing (FcR⁺) and nonbearing (FcR^?) murine splenic lymphocytes was analyzed using the EA rosetting method. In the secondary anti-horse red blood cell (HRBC) response of C3H mice, FcR^? cells showed higher IgM and IgG responses than did FcR⁺ cells. When nylon wool (NW)-purified T cells primed with keyhole limpet hemocyanin (KLH) were fractionated into FcR^? and FcR⁺ T cells, helper activity was proven in the former subset in the cooperation with syngeneic spleen cells primed with dinitrophenylated ascaris extract (DNP-Asc). FcR⁺ T cells showed essentially no helper activity. When FcR^? cells were cultured, neogenesis of FcR⁺ cells was observed on Days 3 to 5. The conversion from FcR^? to FcR⁺ cells was prominent in B cells (40 to 50%), whereas NW-purified nonadherent FcR^? T cells converted poorly (15 to 20%). The converting process was accelerated slightly by mitogens, but was least affected by antigens. To examine the possible contribution of neogeneic FcR⁺ T cells in the helper activity, KLH-primed FcR^? T cells were precultured for 7 days with homologous antigen. The specific helper activity of the cultured T cells proved to be unaffected by the depletion of neogeneic FcR⁺ T cells by EA rosetting. The neogeneic FcR⁺ T cells had no helper activity. It was thus suggested that helper T cells remain in the FcR^? cell fraction and do not convert to the FcR⁺ state during the cooperating process.     

The relative importance of the bone marrow and spleen in the production and dissemination of B lymphocytes.

The relative importance of the bone marrow and spleen in the production of B lymphocytes was investigated in guinea pigs by the combined use of [³H]TdR radio-autography and fluorescent microscopy after the staining of B cells by FITC-F(ab′)₂-goat-anti-guinea pig Ig. Large and small lymphoid cells possess sIg in the marrow and spleen but B cell turnover in the marrow exceeds that in the spleen. That newly generated bone marrow B cells are not derived from an extramyeloid bursa equivalent was demonstrated by the absence of [³H]TdR labeled B cells in tibial marrow 72 hr after [³H]TdR was administered systemically, while the circulation to the hind limbs was occluded. Pulse and chase studies with [³H]TdR showed that large marrow B cells are derived from sIg-negative, proliferating precursors resident in the bone marrow and not from the enlargement of activated small B lymphocytes. The acquisition of [³H]TdR by splenic B cells lagged behind that observed in the marrow. Three days after topical labeling of tibial and femoral bone marrow with [³H]TdR, a substantial proportion of splenic B cells were replaced by cells that had seeded there from the labeled marrow. The studies unequivocally identify the bone marrow as the organ of primary importance in B cell generation and indicate that in the guinea pig rapidly renewed B lymphocytes of the spleen are replaced by lymphocytes recently generated in bone marrow. The rate of replacement of B lymphocytes in the lymph node by cells newly generated in the bone marrow takes place at a slower tempo than in the spleen.     

Accumulation of major histocompatibility complex class II+CD11c− non‐lymphoid cells in the spleen during infection with Plasmodium yoelii is lymphocyte‐dependent

The spleen is the main organ for immune defense during infection with Plasmodium parasites and splenomegaly is one of the major symptoms of such infections. Using a rodent model of Plasmodium yoelii infection, MHC class II⁺CD11c^? non‐T, non‐B cells in the spleen were characterized. Although the proportion of conventional dendritic cells was reduced, that of MHC II⁺CD11c^? non‐T, non‐B cells increased during the course of infection. The increase in this subpopulation was dependent on the presence of lymphocytes. Experiments using Rag‐2^?/? mice with adoptively transferred normal spleen cells indicated that these cells were non‐lymphoid cells; however, their accumulation in the spleen during infection with P. yoelii depended on lymphocytes. Functionally, these MHC II⁺CD11c^? non‐T, non‐B cells were able to produce the proinflammatory cytokines alpha tumor necrosis factor and interleukin‐6 in response to infected red blood cells, but had only a limited ability to activate antigen‐specific CD4⁺ T cells. This study revealed a novel interaction between MHC II⁺CD11c^? non‐lymphoid cells and lymphoid cells in the accumulations of these non‐lymphoid cells in the spleen during infection with P. yoelii.

     

Physiological functions of mitochondrial Na+-Ca2+ exchanger,NCLX, in lymphocytes

Roles of mitochondrial Na⁺-Ca²⁺ exchanger, NCLX, were studied in B lymphocytes such as heterozygous NCLX knockout DT40 cells, NCLX knockdown A20 cells, and native mouse spleen B lymphocytes treated with a NCLX blocker, CGP-37157. Cytosolic Ca²⁺ response to B cell receptor stimulation was impaired in these B lymphocytes, demonstrating importance of mitochondria-ER Ca²⁺ recycling via NCLX and sarco/endoplasmic reticulum Ca²⁺-ATPase SERCA, and interaction with store-operated Ca²⁺ entry. NCLX was also associated with motility and chemotaxis of B lymphocyte. Contrary to B lymphocytes, contribution of NCLX in mouse spleen T lymphocytes was minor.     

Mechanism of histamine-induced inhibition of lymphocyte response to mitogens in mice

Histamine induced, in mice, an inhibition of lymphocyte response to PHA and LPS, at molar concentrations ranging from 10^?3 to 10^?9M. This inhibition occurs as a specific interaction between histamine and T lymphocytes bearing H2-type receptors for this hormone (H + cells) and Ly 2 membrane antigens. Two features of the suppressive activity of this T-cell subpopulation were observed: (i) when histamine is added at the beginning of the culture period with PHA or LPS, it activates the suppressor activity of H + cells which act on the lymphocyte population responding to PHA and LPS; (ii) preincubation of spleen lymphocytes with histamine for 24 hr induces suppressor cells which inhibit the response to PHA, but not to LPS, of syngeneic lymphocytes in a coculture system, and which are radiosensitive. The role of PHA as a second stimulus of histamine-induced suppressor cells, and the relation between these cells and PHA or Con A-induced suppressor cells, are discussed.     

Early postnatal development of the immune system in piglets: the redistribution of T lymphocyte subsets

In this study, we have characterised postnatal changes in T lymphocyte subsets, especially γδ T lymphocytes, in blood, spleen and lymph nodes. Detection was carried out using two-colour flow cytometry and three-colour immunohistochemistry. During ontogeny, there was a significant increase in the total percentage of γδ T cells in the spleen and blood. In the lymph nodes, there were no age-dependent changes in the total percentage of γδ T cells, but the percentage of the γδTCR⁺CD8⁺ subpopulation significantly increased. The tissue distribution of γδTCR⁺CD8⁺ and γδTCR⁺CD8⁻ cells in the lymph nodes is random and not collocated with a particular area of the organ. Furthermore, postnatal development was characterised by an increasing frequency of CD8⁺CD3⁺CD4⁻γδTCR⁻, which was compensated by a decreasing proportion of CD4⁺ lymphocytes. Double positive CD4⁺CD8⁺ lymphocytes were rare during the first month of life and a significant age-dependent increase of these cells was found in all the compartments monitored.     

Na+-dependent,potential-sensitive l-ascorbate transport across brush border membrane vesicles from kidney cortex

l-Ascorbate is taken up into brush border vesicles from kidney cortex of rat, rabbit and guinea pig by an efficient, Na⁺-dependent and potential-sensitive transport process. This uptake shows saturation ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>:0.1–0.3\; mM</mtext>}}$) and is strongly stimulated by low concentrations of N₃^?. Erythorbate (d-isoascorbate) seems to be another, but poorer, substrate of the same transporter.     

Antibody-dependent cellular cytotoxicity effector cells lack Ia antigens.

Surface immunoglobulin (sIg)-positive and sIg-negative subpopulations of macrophage-depleted murine splenic lymphocytes were obtained by Sephadex anti-Fab immunoabsorbent fractionation. These lymphocyte subpopulations were analyzed for the presence of Thy 1 and Ia alloantigens and also for Fc receptors by fluorescence microscopy. Concurrently, these lymphocyte subpopulations were studied for effector cell activity in antibody-dependent cellular cytotoxicity (ADCC). Effector cells mediating ADCC were contained in the sIg-negative lymphocyte subpopulation and sIg-positive lymphocytes did not mediate cytotoxicity. The majority of sIg-positive lymphocytes were found to bear Ia antigens and Fc receptors, and these cell surface structures were associated in that treatment of these cells with anti-Ia sera inhibited binding of complexed immunoglobulin to Fc receptors. In contrast, most sIg-negative, Thy 1-negative lymphocytes lacked Ia Antigens, and the Fc receptors detected on such cells were not blocked by anti-Ia sera. In addition, a small subpopulation of sIg-negative, Ia antigen-positive, Fc receptor-positive lymphocytes was found. Elimination of this subpopulation of Ia antigen-positive cells from sIg-negative lymphocytes, by treatment with anti-Ia serum and complement, did not diminish ADCC effector cell activity in the resultant cell population when compared with untreated sIg-negative lymphocytes. Thus, in murine spleen, nonphagocytic mononuclear cells that lack both sIg and Ia antigens were shown to mediate ADCC.     

Effect of anti-lymphotoxin on cell-mediated cytotoxicity. Evidence for two pathways, one involving lymphotoxin and the other requiring intimate contact between the plasma membranes of killer and target cells.

A rabbit anti-lymphotoxin serum produced against partially purified, antigeninduced, guinea pig lymphotoxin, was used to study the role of lymphotoxin in lymphocyte-mediated cytotoxicity in vitro. The anti-lymphotoxin serum inhibited cytolysis resulting from incubation of ovalbumin-immune guinea pig spleen cells with either mouse (P815 mastocytoma) or guinea pig (line 10 hepatoma) target cells in the presence of soluble ovalbumin. The antiserum also inhibited the cytolysis of ovalbumin-coupled target cells by ovalbumin-immune guinea pig spleen cells. In contrast, the anti-lymphotoxin serum did not inhibit: (a) the lysis of line 10 (strain 2) hepatoma cells by spleen cells from alloimmunized Hartley or strain 13 animals; (b) the lysis of line 10 hepatoma cells by spleen cells from tumor-bearing syngeneic animals; or (c) the lysis of P815-mastocytoma cells by spleen cells from P815-immune guinea pigs. These results support the hypothesis that there are at least two distinct pathways by which immune lymphocytes can destroy target cells in vitro, one which involves secretion of a nonspecific soluble factor, i.e., lymphotoxin, and another which probably requires intimate contact between the plasma membranes of the target and killer cells.     

Lymphocyte-induced angiogenesis factor is produced by L3T4+ murine T lymphocytes,and its production declines with age

Summary Lymphocyte-induced angiogenesis factor (LIA) is a product of T lymphocytes which has been shown to stimulate new vessel formation. Because immune senescence most profoundly affects T lymphocyte functions, we suspected that LIA production would decline with age. An assay for angiogenesis stimulated by allogeneic reaction was performed by injecting spleen cells from young or old donor mice into the skin of irradiated allogeneic recipient mice. The spleen cells from young mice induced a significantly greater number of vessels than did cells from older mice. In additional experiments, spleen cells from young and old animals were treated with a monoclonal antibody GK1.5) directed at the L3T4 antigen on murine T helper lymphocytes. Such treatment significantly reduced the new vessel formation induced by young lymphocytes but had no effect on that induced by lymphocytes from old animals. Studies employing indirect immunofluorescence demonstrated that the proportion of L3T4⁺ cells in the mononuclear fraction of splenocytes was nearly identical in both young and old mice. From these investigations we can conclude that (1) L3T4⁺ lymphocytes are responsible for LIA production, and (2) production, like that of other T lymphokines, declines with age.Supported by NIH grant AG 00332 and VA Merit Award (WBE)     

Cytotoxic effector cell function in organized gut-associated lymphoid tissue (GALT).

Lymphocytes from the organized gut-associated lymphoid tissues (GALT) of adult guinea pigs were examined for surface markers characteristic of T and B lymphocytes and for their capacity to function as effector cells in mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) reactions. GALT lymphocytes formed rosettes with rabbit erythrocytes, a T-cell marker, and underwent proliferative responses in vitro in the presence of phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM). GALT lymphocytes were cytotoxic in vitro for erythrocyte and DBA mastocytoma targets in the presence of PHA. A population of GALT lymphocytes bound aggregated γ-globulin; however, they functioned poorly in ADCC reactions. Thus, organized GALT in the guinea pig contains lymphocytes capable of functioning in T-cell-dependent MICC reactions but either lacks the effector cell population which mediates ADCC or contains an effector cell which functions poorly in ADCC.     

In vivo and in vitro synergistic antitumor effect of interleukin-2-cultured tumor-bearer spleen cells and immune fresh spleen cells

Summary The synergistic antitumor effect of interleukin-2(IL-2)-cultured tumor-bearer spleen cells (cultured lymphocytes) and immune fresh spleen cells was examined. Tumor-bearer cultured lymphocytes were obtained by culturing BALB/c spleen cells from syngeneic MOPC104E-tumor-bearing mice for 11 days with crude IL-2 and a soluble tumor extract. These cultured lymphocytes had weak antitumor activity when transferred i.p. into tumor-bearing mice that had been inoculated i.p. with 10⁵ tumor cells 5 days previously. Immune fresh spleen cells, obtained from mice in complete remission after the treatment with cyclophosphamide, also had weak antitumor activity when transferred at the same schedule. The cultured cells and the fresh cells, mixed together before transfer, significantly augmented the therapeutic effect. At least 1×10⁷ tumor-bearer cultured lymphocytes and 4×10⁷ immune cells were needed for the synergistic effect. A tumor-specific combination was needed for both cultured and fresh cells. The effective subpopulation of tumor-bearer cultured lymphocytes was a cytotoxic one from an Lyt2⁺ precursor, and that of the immune fresh spleen cells was noncytotoxic, Lytl⁺ and Lyt2⁺ T-cells.A similar synergistic effect was also observed during in vitro coculture of tumor-bearer and immune cells. Cytotoxicity, as assessed by the ⁵¹Cr-release test, of tumor-bearer IL-2-cultured lymphocytes was maintained most effectively after 3 or 4 days of culture without IL-2 when the lymphocytes were cocultured with immune fresh spleen cells and tumor cells.     

Immunological properties of Fc receptor on lymphocytes. 5. Suppressive regulation of humoral immune response by Fc receptor bearing B lymphocytes.

High anti-DNP PFC responses to DNP-DE or DNP-KLH were obtained by transferring normal or primed FcR^? B cell fractions into irradiated syngeneic recipients. On the other hand, the FcR⁺ B cell fraction showed a low precursor activity. Trypsinization of the FcR⁺ B cells, to eliminate remaining antigen-antibody complexes on the surface, failed to augment the response in comparison with that of trypsin-untreated FcR⁺ B cells. Therefore, the weak precursor activity of FcR⁺ B cells seemed to be inherent. No synergistic interaction between the FcR⁺ B and precursor FcR^? B cells, to give rise to the maximum PFC response, was observed. On the contrary, the FcR⁺ B cells significantly suppressed the PFC responses of FcR^? B cells. This kind of suppression could be mediated by a factor released from the FcR⁺ B cell, but not from the FcR^? B or original-unrosetted spleen cell fraction. The factor was not attributable to macrophages, because the FcR⁺ B cells isolated from normal spleen cells, of which macrophages were depleted by Sephadex G-10 columns, could produce the factor with the same activity. Stimulation by specific antigen is not necessary for the induction of the factor(s) as well as of the suppressing FcR⁺ B cells. It seems to be necessary to stimulate FcR by antigen-antibody complexes to produce or release this factor.