ConA Response Research Articles

BIOSYNTHETIC METHIONYL-HUMAN GROWTH HORMONE (BMHGH) TREATMENT AFFECTS IMMUNE FUNCTIONS (IF) IN GROWTH HORMONE-DEFICIENT CHILDREN

We have previously reported transient depression of % B cells, Phytohemagglutinin (PHA) responsiveness and T Helper/Suppressor cell (T H/S) ratios in GH-deficient children during treatment (Tx) with pituitary derived human growth hormone (PHGH) (J Pediatr 109:434, 1986). In vitro, incubation of peripheral blood lymphocytes. (PBL) with GH resulted in decreased % B cells. The PBL of children undergoing GH Tx had increased baseline and GH stimulated proliferation. In the current study, we evaluated IF during Tx with BMHGH (kindly provided by Eli Lilly & Co.) in 7 children, ages 4-15, 2 female. Four pts. had been part of the previously cited report. Cell surface markers (SM) and interleukin-2 receptors (IL-2r) were measured by flow cytometry. All pts. had normal baseline IF. Data obtained during 6 months of Tx were analyzed. % B cells decreased significantly at 1 month of Tx (mean change vs baseline −3.9 ± 3.2, p<.05 by two-tailed paired T test) and remained below baseline in most pts. at 3 and 6 months, confirming our in vitro and in vivo findings with PHGH. % T total cells increased (mean change +3.5 ± 3.4, p<.05) at 1 month. There were no other statistically significant changes in SM. Mitogen responses to PHA and concanavalin-A (Con-A) increased in 5/7 and 6/7 pts. at 1 day-1 month of Tx, in agreement with our in vitro data. At 3 months of Tx PHA and Con-A responses decreased in 2/7 and 6/7 children respectively. IL-2r expression was diminished at 1 day-1 month in 6/7 pts. Most of these changes were transient and none was clinically evident. We provide first evidence that BMHGH treatment does affect IF in children. The effects of BMHGH on IF appear to be similar to those of PHGH. These data further support our concept of the presence of a GH-immune network that needs to be considered when treating children with human growth hormone.

L-carnitine enhances lymphocyte mitogenesis in depleted traumatised and infected patients

The influence of L-carnitine on concanavalin (ConA)-induced lymphocyte mitogenesis in vitro was studied in six depleted patients with severe post-operative infections and in eight healthy control subjects. Three patients had received i. v. carnitine supplementation as part of a clinical trial. One patient was studied both before and after i.v. carnitine supplementation. Five patients showed a poor or absent mitogen response compared with the controls. Carnitine markedly augmented the mitogen response in all these patients. In the patient studied before and after i. v. carnitine supplementation, the low initial ConA response increased to values seen in normal individuals. In the lymphocytes from healthy control subjects, no effect of carnitine on activation could be observed. The results demonstrate a stimulatory effect of L-carnitine on lymphocyte mitogenesis in depleted patients with severe post-operative infections.

Mechanism of action of the new immunomodulator LS2616 on T cell responses

Spleen cells from mice treated with LS2616 display a highly increased response to the polyclonal T cell lectin ConA. The total number of splenic T cells, and the relative ratios between L3T4 + and Lyt-2 + T cells were not altered by LS2616 treatment. By dissecting the overall ConA response it was found that the number of ConA-inducible, IL-2 reactive T cells was unaffected, while ConA-induced IL-2 production was enhanced after LS2616 treatment. Spleen cells from LS2616 treated mice, depleted of G10 adherent macrophages (Mφ) and reconstituted with Mφ from untreated mice displayed normal levels of ConA responses. Mφ depleted spleen cells from untreated animals, cocultured with Mφ enriched populations from LS2616 treated animals resulted in an increased ConA response. Furthermore, spleen cells from treated mice were found to be excellent stimulators for alloantigen-induced T cell responses; when used as responders in MLC, however, these cells were comparable to responders from non-treated animals. Taken together the results demonstrate that LS2616 exerts an immunostimulatory effect on Mφ, which indirectly facilitates polyclonal and antigen-specific T cell responses. The possible implications of this observation on various immunoregulatory events are discussed.

Mitogen responsiveness of lymphocytes from the BB/W rat.

The response of BB diabetes-prone (DP) and W-line non-diabetes-prone rats to the T-cell mitogen concanavalin A (ConA) was measured. The W line was a good responder to ConA, whereas the DP was relatively unresponsive. This unresponsiveness could not be reversed with exogenous interleukin 2 (IL-2). The response of DP rats was enhanced by removing adherent cells. To directly test the response of BB T-cells, they were isolated by flow sorting. These experiments demonstrated that BB T-cells could mount a normal ConA response. The normal function of isolated BB T-cells suggested that they were under suppression. Suppressor activity could not be found in the OX8+ population but was found in the DP-adherent cell population. Adherent cells from the W line were not suppressive at the concentrations used. These results showed that the decreased mitogen responsiveness of BB T-cells was not due to an intrinsic T-cell abnormality but was due, in part, to suppression by adherent cells.

Effect of azimexon therapy on host defense parameters and disease-associated symptoms in the acquired immune deficiency syndrome (AIDS) and AIDS-related complex (ARC).

Azimexon, a 2-cyan-aziridinyl immune modulator, was given at a dose of 250 mg/m2/day for 10 days IV to 12 patients with AIDS and 16 with AIDS related complex (ARC). A decrease in total number of AIDS related symptoms from 43 to 24 and in mean number from 2.6 to 1.5 was observed among ARC patients (p less than .01). The most commonly improved symptoms were diarrhea, fatigue, and weight loss with the least frequently improved being lymphadenopathy. The following improvements in immune parameters were observed among ARC patients. DTH to recall antigens improved with an increase in number of positive tests from 35 to 47 and in mean number of positive skin tests from 2.2 on day 0 to 2.9 on day 14 (P less than .05). The geometric mean of the absolute lymphocyte count was 1.395 X 10(3)/microliter on day 0 with a significant increase of 18.0 percent on day 5 (P less than .01) and a 7.7 percent increase on day 21. The geometric mean of the OKT4+ cells on day 0 was 0.250 X 10(3)/microliter with a 33.3 percent increase on day 5 (P less than .07) and a 14.1 percent increase on day 21. T4/T8 ratio increased by 32.7 percent on day 5 (P less than .05) and by 19.4 percent on day 21 from an initial geometric mean of 0.339 X 10(3)/microliter on day 0. The geometric mean of GVH responses increased by 18.2 percent on day 5 (P less than .05) and by 24.0 percent on day 21 (P less than .07) from an initial value of 41.04 mm3. No symptomatic or immunologic improvements were observed among AIDS patients, but rather a significant decrease in mitogenic responses. PHA responses decreased by 70.3 percent on day 5 (P less than .05) and 42.2 percent on day 21 from an initial geometric mean of 4.02 X 10(3) cpm/10(3). Con-A responses decreased by 75.1 percent on day 5 (P less than .05) and increased by 20.3 percent on day 21 from an initial value of 1.14 X 10(3)/10(5) cells. Pretreatment number of absolute OKT4+ cells was the most significant prognostic survival variable. Thus, 8/9 patients with less than 0.10 X 10(3) OKT4+ blood cell/microliter subsequently died as compared to only 1/17 with greater than or equal to 0.10 X 10(3) OKT4+ cells (p less than .001). The only toxic effect of this treatment was mild hemolysis which disappeared upon cessation of treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

The in vitro Response of Turkey Lymphocytes to Steroid Hormones

The in vitro mitogen response of whole blood turkey lymphocytes to various concentrations of steroid hormones was evaluated. Corticosterone (COS) at concentrations between 1 and 80 ng/ml significantly suppressed the proliferative response (3H-thymidine incorporation) to phytohemagglutinin (PHA) and concanavalin A (ConA). Non-mitogen-stimulated (NMS) cells were suppressed at concentrations of COS above 5 ng/ml. Progesterone significantly suppressed NMS cells at concentrations of 80 ng/ml, PHA-stimulated cells at concentrations of 500 ng/ml, and ConA-stimulated cells at concentrations of 1000 ng/ml. beta-Estradiol enhanced the response of NMS cells at concentrations of 500 ng/ml, had no effect on PHA-stimulated cells, and suppressed the response of ConA-stimulated cells at concentrations greater than 500 ng/ml. Testosterone affected only the ConA response, causing suppression at concentrations above 2000 ng/ml. Corticosterone and progesterone caused 80 and 95% suppression, respectively, of the proliferative response to ConA when compared with non-hormone-treated cells. The possible implications of steroid hormone-induced immunosuppression in the pathogenesis of aspergillosis is discussed.

Dual effects of OK-432 on mitogenic response of splenocytes to concanavalin A.

OK-432, a streptococcal preparation, was studied for its effect on the concanavalin A (Con A)-induced mitogenesis of the host spleen cells. When mice were given a single intraperitoneal injection of OK-432, there was a substantial increase in the mitogenic response of splenocytes, whereas multiple injections conversely resulted in a marked reduction of the mitogenic response, when the spleen cells were cultured at high cell densities of over than 5 X 10(5) cells/well. The reduced Con A-responsiveness in the latter was not restored by mixing spleen cells from mice given multiple OK-432 injections with those from normal mice. Moreover, splenic macrophages from OK-432-injected mice exhibited marked inhibitory activity against Con A-mitogenesis of normal splenocytes, while normal splenic macrophages failed to show such an effect. Splenic T cells from OK-432-injected mice also showed an inhibitory activity against Con A-mitogenesis of normal splenocytes and similar activity was also noted in normal splenic T cells. Therefore, the OK-432-spleen cells contain two types of suppressor cells; one is a newly elicited suppressor macrophage and the other is a suppressor T cell supposedly resident also in normal spleen cells. In the OK-432-injected spleen cells, accessory cell function for T cell Con A-mitogenesis was markedly reduced. On the other hand, it was noted that the interleukin 2-producing ability of the OK-432-splenocytes was augmented more than that of normal splenocytes, indicating that multiple OK-432 injections also cause an increase in the helper T cell activity of the host spleen cells.

Interleukin-l-Like Activity Constitutively Generated by Hodgkin Derived Cell Lines: I. Measurement in a Human Lymphocyte Co-stimulator Assay

Culture supernatants (CS) from Hodgkin derived cell lines have previously been shown to contain colony stimulating activity (CSF) for human cord blood cells, fetal bone marrow and fetal liver cells. In this study 3-day CS from four Hodgkin lines (L428, L538, L540, L591) and two sublines (L428KS, L428KSA) were examined for interleukin (IL) activity. None of the tested CS supported, the growth of an IL-2 dependent murine T-cell line, suggesting that the Hodgkin lines do not produce significant amounts of IL-2. When crude 3-day CS from the various lines were assayed for IL-1-activity in the conventional murine thymocyte co-stimulator assay no or only borderline IL-1-activity was detectable. However, concentrated CS from L428KS exhibited IL-1-activity also in this assay as did lipopolysaccharide (LPS) induced human IL-1. Surprisingly, crude 3-day CS from all Hodgkin cell lines were capable of fully replacing the accessory cell requirement in ConA-induced lymphoproliferation assays of heavily monocyte-depleted human blood lymphocytes. The monocyte-depleted lymphocyte populations were obtained by 1 × g sedimentation at a sedimentation rate of 30.2 to 38.8 mm/ hr (fraction IIIa and IIIb). These cells responded poorly to the T-cell mitogen ConA at 10 υg/ ml and produced no IL-2. Addition of irradiated, autologous monocytes or of CS from the various Hodgkin cell lines quantitatively restored the ConA responsiveness and induced significant IL-2 production in the monocyte-depleted lymphocyte population, suggesting that Hodgkin lines constitutively secrete IL-1 or IL-1-like activity. A preliminary biochemical characterization (heat and pH stability, molecular weight range of 13-24 KD) supports the notion that the accessory cell replacing activity present in CS of Hodgkin cell lines is a type of human IL-1.

Asbestos-induced alterations of human lymphoid cell mitogenic responses

Using mitogenic assays, we have investigated the short term effects of two asbestos (amosite and chrysotile) fibers on lymphocyte functions in vitro. These oppositely charged fibers produced different alterations in mitogenesis. The blastogenic responses of concanavalin-A (Con-A) and pokeweed mitogen stimulated human peripheral blood mononuclear cells (PBMN) were significantly increased by the inclusion of 6 μg of chrysotile to the culture media. Amosite fibers proved to be inhibitory in all tests. When PBMN were depleted of monocytes, asbestos-related alterations of Con-A responsiveness were unchanged among the remaining cells. However, the addition of chrysotile to phytohemagglutinin (PHA) cultures resulted in a significant increase of the mitogenic response. When PBMN were enriched for T lymphocytes, and again cultured with the mitogens and fibers, the Con-A response still displayed impressive enhancement with chrysotile. In contrast to an intact PBMN population, PHA-induced blastogenesis among these T-enriched lymphocytes was significantly elevated. These experiments demonstrate that asbestos can induce significant changes in the functional integrity of PBMN following a relatively short exposure time in culture.

Mitogen response of peripheral blood and splenic lymphocytes and effect of 2-mercaptoethanol in tumor-bearing mice

A murine osteosarcoma in which the number of tumor cells can be continually monitored by measuring the circulating plasma alkaline phosphatase levels was used to determine the effect of tumor burden on peripheral blood and splenic lymphocyte response to mitogens. In animals with tumors of different sizes, the pattern of response of the peripheral blood lymphocytes (PBL) to mitogens is different from that of splenic lymphocytes. PBL response to ConA, PHA, and LPS was initially depressed, but response to PHA and LPS recovered later, as the tumor burden exceeded 6%. However, the recovery of LPS response was not consistent, in that recovery was not seen when the tumor burden was 5%–6%. Response to ConA remained depressed. Splenic lymphocytes showed progressive decline of PHA response. Treatment of tumor-bearing mice with 2-mercaptoethanol (2-ME) restored the ConA response of PBL in 56% of mice. Treatment with 2-ME did not restore PBL response to PHA or LPS.

AN IN VIVO AND IN VITRO STUDY OF THE CYCLOPHOSPHAMIDE‐INDUCED ENHANCEMENT OF CONTACT SENSITIVITY TO 2,4‐DINITRO‐1‐CHLOROBENZENE (DNCB)

ABSTRACT Enhancement of DNCB contact sensitivity (CS) reactions by cyclophosphamide (Cy) was observed in guinea pigs sensitized by painting or footpad injection of Freund's incomplete adjuvant, in which immunologic suppression of CS had been induced, but not in animals sensitized by footpad injection of Freund's complete adjuvant. The latter developed markedly strong CS reactions. The effects of Cy on in vivo skin reactions and on in vitro endogenous and antigen‐induced DNA synthesis by the draining lymph node cells in animals sensitized topically with DNCB were studied on various days after sensitization. Cy treatment 3 days before sensitization augmented CS reactions. This augmentative effect was found from day 5 to day 21. Cy treatment enhanced in vitro antigen‐induced DNA synthesis as well as endogenous DNA synthesis by the draining lymph node cells. Cy‐sensitive cells and their progeny may be involved in suppression of the in vitro specific stimulation of the primed cells as well as endogenous cell proliferation for priming. The effect of DNCB‐sensitization on the in vitro concanavalin A (ConA) dose‐response of the spleen cells and lymph node cells was studied. By day 7 after sensitization, the stimulation of the spleen cells by low doses of ConA had decreased, but the responses to high doses of mitogen had increased. This bidirectional effect of DNCB‐sensitization on ConA responses was discussed.

Autologous mixed lymphocyte culture responder T cells are in the concanavalin A reactive subpopulation and separate from alloreactive cells

We have characterized the T cells which respond to non-T cells in the autologous mixed lymphocyte culture (AMLC) reaction. Lymphocytes were separated by discontinuous gradient fractionation, and populations were eliminated by treating either concanavalin A (Con-A) or AMLC-responding T cells with Budr and subsequent light exposure. The results show that AMLC-responding T cells occur in gradient fractions showing maximal Con-A responsiveness and that deletion of Con-A responsive cells by Budr followed by light exposure also deletes the AMLC-responding T cells. Con-A and MLC responses of cells remaining after Budr plus light treatment of AMLC were vigorous, although AMLC responses were abrogated. AMLC responses after Budr + light treatment of MLC-activated T cells were comparable to those in control cultures but MLC responses were decreased as expected. Taken together, these results indicate that the T cells which respond in AMLC are a subset of the Con-A responsive T-cell population and that they are separate from MLC-responding T cells.

Heterogeneous Mechanisms of Impaired Lymphocyte Responses in Non-Hodgkin’s Lymphoma

AbstractPeripheral blood mononuclear cells (PBMC) from 18 untreated patients with non-Hodgkin’s lymphoma (NHL) were studied to characterize the cellular mechanisms contributing to impaired in vitro lymphocyte responses after stimulation by the mitogen conconavalin A (Con-A). In vitro reactivity was quantitated by the 3H-thymidine incorporation in response to an optimal dose of Con-A. All patients demonstrated impaired in vitro reactivities compared to normal controls. These in vitro impairments were partially reversible since patient’s cells precultured in media alone for 3 days demonstrated enhanced Con-A responses. In greater than half of the patients, the hyporeactive PBMC suppressed the enhanced reactivities of autologous precultured PBMC when assayed in cocultures. Suppressor activity was detected mainly in those untreated patients presenting with either constitutional symptoms or diffuse histology and in general was not marked compared to the severity of impairments. Adherent monocytes were shown to participate in the suppression of autologous lymphocyte reactivity but only appeared partially responsible for the in vitro impairments. In those patients lacking detectable suppressive activity, preculturing also enhanced Con-A reactivities and was compatible with the presence of a reversible, inhibitory mechanism differing from active suppression. Many patients’ hyporeactive PBMC, however, failed to demonstrate normal responses after preculturing. This failure could not be directly attributed to aberrant regulatory populations, but rather appeared to possibly represent an additional intrinsic impairment of potentially reactive populations.

Heterogeneous mechanisms of imparied lymphocyte responses in non- Hodgkin's lymphoma

Peripheral blood mononuclear cells (PBMC) from 18 untreated patients with non-Hodgkin's lymphoma (NHL) were studied to characterize the cellular mechanisms contributing to impaired in vitro lymphocyte responses after stimulation by the mitogen conconavalin A (Con-A). In vitro reactivity was quantitated by the 3H-thymidine incorporation in response to an optimal dose of Con-A. All patients demonstrated impaired in vitro reactivities compared to normal controls. These in vitro impairments were partially reversible since patient's cells precultured in media alone for 3 days demonstrated enhanced Con-A responses. In greater than half of the patients, the hyporeactive PBMC suppressed the enhanced reactivities of autologous precultured PBMC when assayed in cocultures. Suppressor activity was detected mainly in those untreated patients presenting with either constitutional symptoms or diffuse histology and in general was not marked compared to the severity of impairments. Adherent monocytes were shown to participate in the suppression of autologous lymphocyte reactivity but only appeared partially responsible for the in vitro impairments. In those patients lacking detectable suppressive activity, preculturing also enhanced Con-A reactivities and was compatible with the presence of a reversible, inhibitory mechanism differing from active suppression. Many patients' hyporeactive PBMC, however, failed to demonstrate normal responses after preculturing. This failure could not be directly attributed to aberrant regulatory populations, but rather appeared to possibly represent an additional intrinsic impairment of potentially reactive populations.

Increased radiosensitivity of a subpopulation ot T-lymphocyte progenitors from patients with Fanconi's anemia

In vitro radiation survival of peripheral blood T lymphocytes was studied in 15 clinically normal adults and 4 patients with Fanconi's anemia. Tritiated thymidine incorporation in a whole blood lymphocyte stimulation test (LST) and a newly developed whole blood T-lymphocyte colony assay were used to measure lymphocyte blastogenesis and colony formation in response to phytohemagglutinin (PHA) or concanavalin-A (Con-A) stimulation. Lymphocyte colony formation was found to be consistently more sensitive than the LST for detection of low-level radiation effects using both normal cells and lymphocytes from Fanconi's anemia patients. Lymphocytes from patients with Fanconi's anemia were significantly more sensitive to in vitro x-irradiation than lymphocytes from clinically normal individuals as measured by their ability to divide when stimulated by PHA in the LST (patients, D37 = 198 R; normals, D37 = 309 R, p = 0.057) and colony formation assay (patients, D37 = 53 R; normals, D37 = 109 R, p = 0.016). No significant difference in the radiosensitivity of the Con-A response was observed between the two groups. The PHA-responsive T-lymphocyte subpopulation in Fanconi's anemia patients appears to be intrinsically defective. The nature of this defect, significance in the disease process, and relevancy of these findings to the establishment of radiation protection standards are discussed.

Increased Radiosensitivity of a Subpopulation of T-Lymphocyte Progenitors From Patients With Fanconi’s Anemia

AbstractIn vitro radiation survival of peripheral blood T lymphocytes was studied in 15 clinically normal adults and 4 patients with Fanconi’s anemia. Tritiated thymidine incorporation in a whole blood lymphocyte stimulation test (LST) and a newly developed whole blood T-lymphocyte colony assay were used to measure lymphocyte blastogenesis and colony formation in response to phytohemagglutinin (PHA) or concanavalin-A (Con-A) stimulation. Lymphocyte colony formation was found to be consistently more sensitive than the LST for detection of low-level radiation effects using both normal cells and lymphocytes from Fanconi’s anemia patients. Lymphocytes from patients with Fanconi’s anemia were significantly more sensitive to in vitro x-irradiation than lymphocytes from clinically normal individuals as measured by their ability to divide when stimulated by PHA in the LST (patients, D37 = 198 R; normals, D37 = 309 R, p = 0.057) and colony formation assay (patients, D37 = 53 R; normals, D37 = 109 R, p = 0.016). No significant difference in the radiosensitivity of the Con-A response was observed between the two groups. The PHA-responsive T-lymphocyte subpopulation in Fanconi’s anemia patients appears to be intrinsically defective. The nature of this defect, significance in the disease process, and relevancy of these findings to the establishment of radiation protection standards are discussed.

Inhibition of normal allogeneic lymphocyte mitogenesis by a factor released from human tumor cells in culture

Human tumor and normal cell lines in culture were examined for the release of factors capable of inhibiting lymphocyte blastogenesis. Supernatants from tumor cell cultures of melanoma, carcinoma (lung, colon, breast), sarcoma, and normal fibroblasts inhibited normal lymphocyte response to PHA. Only supernatants from the tumor cell lines C1 (colon carcinoma), 734B (breast carcinoma), and 231 (breast carcinoma) were found to inhibit both PHA and ConA responses significantly. The two breast carcinoma cell lines, 734B and 231, which also were capable of inhibiting lymphocyte responses to PPD and alloantigens, were investigated further. The inhibition of lymphocyte blastogenesis caused by the supernatant of these two cell lines could not be overcome by the addition of added mitogen. Further experiments showed that the inhibition was not due to nutrient deficiencies and the supernatants were not directly toxic to the lymphocyte cultures as judged by trypan blue exclusion.

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

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

Recovery of immune competence after tumour resection in mice: correlation with loss of suppressor elements

Changes in the immune competence and levels of suppressore elements were assessed by mitogen stimulation and in vitro antibody production, after resection of a transplantable sarcoma. Spleen cells from tumour-resected animals were found to have depressed responses to conA as well as to the antigens SRBC and DNP-LPS. This inability to respond was gradually overcome and, by Day 21 after resection, spleen cell competence had returned to normal levels. Suppressor cells isolated from the spleens of tumour-resected animals were capable of suppressing the conA response and PFC response of normal syngeneic spleen cells in vitro. The ability to suppress the conA response of normal cells disappeared by Day 1 after resection, while the ability to suppress the anti-SRBC and anti-DNP PFC response of normal cells disappeared by Day 8 and Day 14 respectively. Serum from tumour-resected mice was also found to be suppressive to the conA response of normal spleen cells. The inhibitory material responsible for suppression eluted with the Ig-containing fraction on Sephadex G-150. This inhibitory material gradually disappeared from the serum of tumour-resected mice and was no longer apparent by Day 14. Therefore, it appeared that the return of normal lymphocyte function after tumour-resection was concomitant with the disappearance of splenic suppressor cells and suppressive serum factor.

Selective responses of mouse T lymphocytes to different T mitogens and in mixed lymphocyte culture induced cytolysis reaction

CBA spleen T lymphocytes were stimulated by the T mitogens concanavalin-A (Con-A), phytohemagglutinin (PHA), and leukoagglutinin (LA). On the 2nd to 3rd culture day the activated cells (blasts) were separated from the nonactivated cells (lymphocytes) by 1 g velocity sedimentation. The lymphocytes which were not activated during the primary culture (lymphocyte fraction from the velocity sedimentation) were then stimulated by the same mitogens or in one-way MLC to DBA/2 m, and tested for relevant target lysis after MLC stimulation. Primary stimulation with Con-A abolished the responses to Con-A, to PHA, and to LA, whereas primary stimulation with PHA or with LA abolished the responses to these mitogens but left behind a considerable Con-A response. Stimulation with any one of the listed T mitogens did not significantly affect the MLC responses. While primary stimulation with Con-A abolished the relevant target cell lysis after MLC stimulation, primary stimulation with PHA or with LA reduced it only slightly. Assuming that the various mitogens stimulate separate subpopulations of T cells, the results seem to indicate that the Con-A-responsive population includes the PHA- and LA-responsive populations but not the MLC-responsive population. It also appears that the T cells generated to killer cells during MLC are mainly confined to the concanavalin-responsive population.