Depletion Of Lymphocyte Subsets Research Articles

Intraperitoneal administration of a modified vaccinia virus Ankara confers single-chain interleukin-12 expression to the omentum and achieves immune-mediated efficacy against peritoneal carcinomatosis

BackgroundPeritoneal carcinomatosis is an advanced stage of cancer in which the disease has spread to the peritoneal cavity. In order to restore antitumor immunity subverted by tumor cells in this...

The Effects of Different Induction Regimes on Serial Lymphocyte Subsets in Kidney Transplant Recipients: A Single Tertiary Center Experience

Immunosuppressive therapy is the backbone of kidney transplantation in preventing acute rejection. T-cell depletion after doses of thymoglobulin is dose-dependent, as are their side effects. At the same time, basiliximab and other maintenance immunosuppressive drugs act at different signals on T lymphocytes. Therefore, studying the pattern of lymphocyte subset depletion depending on the induction regime given at transplantation could be an added tool in managing post-transplant recipients. This prospective observational study recruited kidney transplant recipients from August 2019 through April 2021 at the University of Malaya Medical Centre. Blood tests for lymphocyte subsets were taken at pre-transplant, 1 week, 1 month, 3 months, and 6 months post-transplantation. At transplantation, recipients received either basiliximab, low-dose thymoglobulin (cumulative dose: 1.5 mg/kg), or standard-dose thymoglobulin (cumulative dose: 5 mg/kg). A total of 39 patients were recruited: 38.5% received basiliximab (15 of 39), 15.4% received low-dose thymoglobulin (6 of 39), and 46.2% received standard-dose thymoglobulin (18 of 39). Absolute lymphocyte counts 1 week post-transplantation were 1.5 ± 0.84×109/L for basiliximab, 0.7 ± 0.57×109/L for low-dose thymoglobulin, and 0.1 ± 0.08×109/L for standard-dose thymoglobulin (P < .001). The CD4+ and CD8+ counts were severely depleted in the standard-dose thymoglobulin group, with a statistically significant differenceup to 6 months post-transplantation. In the low-dose thymoglobulin group, the CD4+ and CD8+ counts were depleted at 1 week post-transplantation and recovered at 1 month post-transplantation. There was no difference in allograft function and incidence of allograft rejection across groups. The effects on lymphocyte counts, CD4+ and CD8+, vary depending on the type and dose of induction immunosuppression. This could be a guiding tool in managing immunosuppression post-transplantation depending on the patient's immunologic risk.

Immune Profile in Patients With COVID-19: Lymphocytes Exhaustion Markers in Relationship to Clinical Outcome.

The velocity of the COVID-19 pandemic spread and the variable severity of the disease course has forced scientists to search for potential predictors of the disease outcome. We examined various immune parameters including the markers of immune cells exhaustion and activation in 21 patients with COVID-19 disease hospitalised in our hospital during the first wave of the COVID-19 pandemic in Slovakia. The results showed significant progressive lymphopenia and depletion of lymphocyte subsets (CD3+, CD4+, CD8+ and CD19+) in correlation to the disease severity. Clinical recovery was associated with significant increase in CD3+ and CD3+CD4+ T-cells. Most of our patients had eosinopenia on admission, although no significant differences were seen among groups with different disease severity. Non-survivors, when compared to survivors, had significantly increased expression of PD-1 on CD4+ and CD8+ cells, but no significant difference in Tim-3 expression was observed, what suggests possible reversibility of immune paralysis in the most severe group of patients. During recovery, the expression of Tim-3 on both CD3+CD4+ and CD3+CD8+ cells significantly decreased. Moreover, patients with fatal outcome had significantly higher proportion of CD38+CD8+ cells and lower proportion of CD38+HLA-DR+CD8+ cells on admission. Clinical recovery was associated with significant decrease of proportion of CD38+CD8+ cells. The highest AUC values within univariate and multivariate logistic regression were achieved for expression of CD38 on CD8+ cells and expression of PD1 on CD4+ cells alone or combined, what suggests, that these parameters could be used as potential biomarkers of poor outcome. The assessment of immune markers could help in predicting outcome and disease severity in COVID-19 patients. Our observations suggest, that apart from the degree of depletion of total lymphocytes and lymphocytes subsets, increased expression of CD38 on CD3+CD8+ cells alone or combined with increased expression of PD-1 on CD3+CD4+ cells, should be regarded as a risk factor of an unfavourable outcome in COVID-19 patients. Increased expression of PD-1 in the absence of an increased expression of Tim-3 on CD3+CD4+ and CD3+CD8+ cells suggests potential reversibility of ongoing immune paralysis in patients with the most severe course of COVID-19.

The immunostimulatory effect of lenalidomide on NK-cell function is profoundly inhibited by concurrent dexamethasone therapy

AbstractLenalidomide combined with dexamethasone is an effective treatment for refractory/relapsed multiple myeloma (MM). Lenalidomide stimulates natural killer (NK) cells and enhances antitumor responses. We assessed NK cell number and function in 25 patients with MM participating in a clinical trial of lenalidomide and dexamethasone. NK cell numbers increased from a mean of 2.20 ± 0.05 × 105/mL (baseline) to a mean of 3.90 ± 0.03 × 105/mL (cycle 6; P = .05); however, in vitro NK-cell–mediated cytotoxicity decreased from 48.9% ± 6.8% to 27.6% ± 5.1% (P = .0028) and could not be rescued by lenalidomide retreatment. Lenalidomide increased normal donor NK-cell cytotoxicity in vitro from 38.5% to 53.3%, but this was completely abrogated by dexamethasone. Dexamethasone suppression of NK cell–mediated cytotoxicity was partially reversed by a 3-day washout, but these cells remained refractory to lenalidomide-induced enhanced function. Lymphocyte subset depletion experiments revealed that lenalidomide's enhancement of NK cell–mediated cytotoxicity was mediated by CD4+ T-cell production of interleukin 2 and that dexamethasone acted by suppressing interleukin-2 production. Similarly, the reduced ability of NK cells from patients with MM to respond to lenalidomide was also due to impaired CD4 T-cell function. Our findings indicate that lenalidomide immunostimulatory effects on patient NK cells are severely blunted by concurrent dexamethasone administration.

A murine model of severe immune thrombocytopenia is induced by antibody- and CD8+ T cell–mediated responses that are differentially sensitive to therapy

AbstractImmune thrombocytopenia (ITP) is a bleeding disorder characterized by antibody-opsonized platelets being prematurely destroyed in the spleen, although some patients with ITP may have a cell-mediated form of thrombocytopenia. Although several animal models of ITP have been developed, few mimic primary chronic ITP nor have any shown cell-mediated platelet destruction. To create this type of model, splenocytes from CD61 knockout mice immunized against CD61+ platelets were transferred into severe combined immunodeficient (SCID) (CD61+) mouse recipients, and their platelet counts and phenotypes were observed. As few as 5 × 104 splenocytes induced a significant thrombocytopenia and bleeding mortality (80%) in recipients within 3 weeks after transfer. Depletion of lymphocyte subsets before transfer showed that the splenocyte's ability to induce thrombocytopenia and bleeding completely depended on CD4+ T helper cells and that both CD19+ B cell (antibody)– and CD8+ T cell (cell)–mediated effector mechanisms were responsible. Treatment of the SCID mouse recipients with intravenous γ-globulins raised platelet counts and completely prevented bleeding mortality induced by antibody-mediated effector mechanisms but did not affect cell-mediated disease. This novel model not only shows both antibody- and cell-mediated ITP and bleeding but also suggests that these 2 effector mechanisms have a differential response to therapy.

Delivery of NKG2D Ligand Using an Anti-HER2 Antibody-NKG2D Ligand Fusion Protein Results in an Enhanced Innate and Adaptive Anti-Tumor Response.

Abstract Background: NKG2D ligands activate via the NKG2D receptor expressed on effector cells belonging to both the innate (NK) and adaptive immune systems (CD8+ T cells). Over-expression of NKG2D ligand on the surface of tumor cells leads to tumor regression in several murine models. We reasoned that antibody targeting of NKG2D ligand to tumor cells could also be used to evoke an enhanced anti-tumor immune response.Methods: We fused the murine NKG2D ligand, Rae-1β, to the 3' end of an anti-HER2 IgG3 containing an intact Fc domain (anti-HER2 IgG3-Rae-1β) to target NKG2D ligand to HER2+ breast tumor cells. The cytotoxicity of the fusion proteins was tested in vitro in direct and indirect assays with the murine NK cell line, KY-2. Anti-tumor efficacy was examined in a murine mammary tumor model.Results: Anti-HER2 IgG3-Rae-1β fusion protein bound to both the HER2 antigen, and to NKG2D as determined by flow cytometry. Anti-HER2 IgG3-Rae-1β fusion induced enhanced cytotoxicity of NK effectors against HER2 expressing tumor cells in vitro. EMT6-HER2 bearing BALB/c mice treated with anti-HER2 IgG3-Rae-1β showed markedly decreased tumor growth relative to anti-HER2 IgG3 or PBS injected control mice. Treatment of mice that were implanted on one flank with EMT6 and contralaterally implanted with EMT6-HER2, resulted in prompt regression of EMT6-HER2, and delayed regression of EMT6, suggesting “epitope spreading.” To examine the involvement of NK, CD4, or CD8 cells in the growth inhibition of the Rae-1β fusion protein targeted tumors, we performed lymphocyte subset depletion studies. Mice depleted of either NK cells or CD8+ T cells showed decreased inhibition of tumor growth following treatment with anti-HER2 IgG3-Rae-1β, compared to control mice. These results suggest that NK cells and CD8+ cells play an important role in the anti-tumor activity of anti-HER2 antibody-Rae-1β fusion. Anti-HER2 IgG3-Rae-1β also inhibited growth of EMT6-HER2 in FcγR-/- mice, indicating activity of the fusion in the absence of FcR interaction. No anti-tumor efficacy of anti-HER2 IgG3-Rae-1β was observed in IFNγ-/- mice.To determine if anti-HER2 antibody-Rae-1β fusion led to priming of an adaptive response, twelve mice that had previously rejected EMT6-HER2 tumors following treatment with anti-HER2 IgG3-Rae-1γ, were rechallenged with either EMT6-HER2, or with parental EMT6. Five of six mice rechallenged with EMT6-HER2 and five of six mice rechallenged with EMT6 showed no evidence of tumor growth at day 25, while both EMT6 and EMT6-HER2 grew rapidly in six of six control mice, respectively.Conclusion: Antibody-NKG2D ligand fusion proteins administration leads to an enhanced anti-tumor innate and adaptive immune response. The immune response evoked by targeting of the fusion to HER2, a single surface antigen, encompassed additional non-targeted antigens. Antibody-NKG2D ligand fusion proteins could potentially be used to treat other malignancies by substituting alternate tumor specificities. Murine NKG2D ligand in the antibody fusion molecules could be replaced with human NKG2D ligands for testing in man. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6102.

Antibody- and Cell-Mediated Immune Thrombocytopenia Are Differentially Sensitive to Intravenous Gammaglobulin Therapy

Immune thrombocytopenic purpura (ITP) is a bleeding disorder characterized by IgG autoantibody-opsonized platelets prematurely being destroyed in the spleen although recent evidence suggests that thrombocytopenia in perhaps as many as 40% of patients with ITP can be mediated by CD8+ T cells. Although several animal models of immune thrombocytopenia have been developed, few are induced by platelet-specific autoimmune mechanisms nor have any demonstrated cell-mediated platelet destruction. We developed a murine model of ITP by first immunizaing GPIIIa (CD61) knockout (KO) mice against wildtype (WT) CD61+ platelet transfusions and subsequently transferring their splenocytes (5×104 cells/transfer) intraperitonealy into irradiated SCID mouse recipients. The SCID mice developed significant bleeding mortality and thrombocytopenia within 2 weeks post-transfer. Lower doses (<104) of immune splenocytes transferred induced thrombocytopenia in the SCID mouse recipients with no mortality. Depletion of lymphocyte subsets in the splenocytes before transfer showed that both CD19+ B cell (antibody)- and CD8+ T cell (cell)-mediated thrombocytopenia existed and that bleeding mortality was particularly associated with antibody-mediated thrombocytopenia. Treatment of the SCID mouse recipients bi-weekly with 2g/kg intravenous gammaglobulins (IVIg) demonstrated that IVIg was beneficial in eliminating bleeding mortality and thrombocytopenia only in the recipients displaying the antibody-mediated form of ITP. Cell-mediated bleeding mortality and thombocytopenia was completely resistant to IVIg therapy. This model suggests a potential reason why some patients with ITP fail IVIg therapy and may aid in the development of new therapeutics for individuals inflicted with cell mediated thrombocytopenia.

An epigenetic vaccine model active in the prevention and treatment of melanoma

BackgroundNumerous immune genes are epigenetically silenced in tumor cells and agents such as histone deacetylase inhibitors (HDACi), which reverse these effects, could potentially be used to develop therapeutic vaccines. The conversion of cancer cells to antigen presenting cells (APCs) by HDACi treatment could potentially provide an additional pathway, together with cross-presentation of tumor antigens by host APCs, to establish tumor immunity.MethodsHDACi-treated B16 melanoma cells were used in a murine vaccine model, lymphocyte subset depletion, ELISpot and Cytotoxicity assays were employed to evaluate immunity. Antigen presentation assays, vaccination with isolated apoptotic preparations and tumorigenesis in MHC-deficient mice and radiation chimeras were performed to elucidate the mechanisms of vaccine-induced immunity.ResultsHDACi treatment enhanced the expression of MHC class II, CD40 and B7-1/2 on B16 cells and vaccination with HDACi-treated melanoma cells elicited tumor specific immunity in both prevention and treatment models. Cytotoxic and IFN-γ-producing cells were identified in splenocytes and CD4+, CD8+ T cells and NK cells were all involved in the induction of immunity. Apoptotic cells derived from HDACi treatments, but not H2O2, significantly enhanced the effectiveness of the vaccine. HDACi-treated B16 cells become APCs in vitro and studies in chimeras defective in cross presentation demonstrate direct presentation in vivo and short-term but not memory responses and long-term immunity.ConclusionThe efficacy of this vaccine derives mainly from cross-presentation which is enhanced by HDACi-induced apoptosis. Additionally, epigenetic activation of immune genes may contribute to direct antigen presentation by tumor cells. Epigenetically altered cancer cells should be further explored as a vaccine strategy.

A MUC1/IL-18 DNA vaccine induces anti-tumor immunity and increased survival in MUC1 transgenic mice

MUC1 (mucin 1) is a tumor-associated antigen that is overexpressed in many adenocarcinomas. Active immunotherapy targeting tumors expressing MUC1 could have great treatment value. MUC1 DNA vaccines were evaluated in MUC1 transgenic (MUC1.Tg) mice challenged with MC38/MUC1 + tumor cells. Vaccination with MUC1 plasmid DNA (pMUC1) alone was insufficient to induce tumor protection. However, co-administration of pMUC1 with a plasmid encoding murine interleukin-18 (pmuIL-18) resulted in significant tumor protection and survival after tumor challenge. Protection was durable in the absence of additional vaccination, as demonstrated by continued protection of vaccinated mice following tumor rechallenge. Mice surviving challenges with MC38/MUC1 + cells showed significant protection after challenge with MUC1 − MC38 tumor cells, suggesting that these mice had developed immune responses to epitopes shared between the tumor cell lines. Antibody-mediated depletion of lymphocyte subsets demonstrated that protection was due largely to CD4 + T cells. This work demonstrates that a naked DNA vaccine can break tolerance to MUC1 and induce an immune response capable of mediating both significant protection from tumor challenge and increased survival.

Long‐term treatment of pigs with low doses of monoclonal antibodies against porcine CD4 and CD8 antigens

In vivo depletion of lymphocyte subsets allows investigation of the role of specific subsets in protective immunity. In the present study we evaluated the effects of long-term, low-dose treatment with murine monoclonal antibodies (mAbs) against porcine CD4 and CD8 surface antigens on lymphocyte subsets in pigs. Four-week-old pigs were treated by intramuscular injections of hybridoma cell culture supernatants containing anti-CD mAbs twice a week for a period of 5 weeks. The immunomodulatory effects of the treatments were assessed by flow cytometry (FCM) analysis of peripheral blood lymphocytes. Treatment with the anti-CD4 mAb almost completely eliminated the CD4(+) T-cell subset from the circulation after 2 weeks of therapy. This depletion persisted until the end of the experimental period 5 weeks after initiated therapy. Treatment with the anti-CD8 mAb was less effective, reducing the CD8(+) T-cell subset in peripheral blood by approximately 50% of the initial level after 3 weeks of therapy. Further, the anti-CD8 mAb-treated pigs showed a parallel increase in the CD4(+) T-cell subset from day 7. Two-colour FCM analysis indicated that a shift in phenotype from single-positive CD4(+)/CD8(-) to double-positive CD4(+)/CD8(+) T-cells might have occurred in these pigs. In the present experiment we demonstrated specific modulation of the peripheral blood T-lymphocyte population in pigs with continuous low-dose injections of specific mAb. The ability to modulate individual T-cell subsets should provide a method to elucidate their functionality in protection against infectious disease.

Long-term in vivo depletion of functional CD4+ T lymphocytes from calves requires both thymectomy and anti-CD4 monoclonal antibody treatment.

In vivo depletion of lymphocyte subsets is a direct approach used for dissection of the mechanisms of protective immunity. Long-term in vivo depletion of bovine T lymphocyte subpopulations with monoclonal antibody (mAb) treatment alone has been difficult to achieve. The objective of this study was to determine whether both thymectomy and anti-CD4 mAb treatment would optimize long-term in vivo depletion of functional bovine CD4+ T lymphocytes. Calves were thymectomized and treated with high doses of anti-CD4 mAb (approximately 5 mg/kg) over 4 days followed by subsequent lower doses (approximately 0.3 mg/kg) administered twice weekly for an additional 7 weeks. Depletion of CD4+ T lymphocytes from blood, spleen and peripheral lymph nodes was significantly improved in thymectomized calves compared to thymus-intact anti-CD4 mAb-treated calves. Significant differences in percentages of CD4+ T lymphocytes between thymectomized and thymus-intact calves were sustained for the duration of the 8-week study. Depletion of CD4+ T lymphocytes from thymectomized calves resulted in complete abrogation of lymphoproliferative responses to ovalbumin. In addition, thymectomized calves treated with anti-CD4 mAb had significantly reduced immunoglobulin G1 and no detectable immunoglobulin G2 ovalbumin-specific antibody responses compared to thymus-intact anti-CD4 mAb-treated calves. The results of this study demonstrate that both thymectomy and treatment with anti-CD4 mAb are required for long-term in vivo depletion of functional bovine CD4+ T lymphocytes.

Use of mRNA differential display to study the action of lymphocyte subsets in vivo and application to a murine model of herpes simplex virus infection

Depletion of lymphocyte subsets in vivo using monoclonal antibodies against cell surface markers has helped to define the roles for these subsets in many immune processes. However, in some cases the mechanisms through which these lymphocytes act remain partially elucidated or completely unknown. A new approach to these biological problems is the use of transcriptional analyses to find mRNAs whose abundance in tissues is altered by depletion of lymphocyte subsets. We have verified the use mRNA differential display (DD) for this purpose and applied it in a study of CD8 + lymphocyte mediated clearance of herpes simplex virus (HSV) from the nervous systems of experimentally infected mice. The results of the differential displays and characterisation of a large mRNA identified using this strategy are presented.

In vivo T-cell subset depletion suggests that CD4 + T-cells and a humoral immune response are important for the elimination of orf virus from the skin of sheep

In vivo lymphocyte subset depletion offers a unique opportunity to study the roles of different cellular components of the immune system of sheep during infection with orf virus. Lambs were depleted of specific lymphocyte subsets by the intravenous administration of monoclonal antibodies against ovine lymphocyte surface markers and then challenged with orf virus. The skin lesions that developed were scored visually as to their severity. Blood samples were collected to monitor the lymphocyte depletions and to measure orf-virus-specific antibody levels. Skin biopsies were collected from the lesion site and studied to determine the course of the infection and the presence of various cell types and orf virus. All the sheep developed orf virus lesions after infection. All three of the CD4-depleted lambs were unable to clear virus from their skin and did not have an antibody response to the virus. Virus was also detected in the skin of one each of the three CD8-depleted, WC1-depleted and control sheep on the final day of the trial. CD8 + lymphocytes did not appear to be essential for viral clearance later in the infection. Depletion of the majority of γδ + T-cells did not affect the outcome of orf virus infection. In sheep with high orf-virus-specific antibody titres at the time of infection, orf lesions healed faster than lesions in sheep with low antibody levels, and this occurred regardless of the lymphocyte depletion status of the animals. This study suggests that the presence of CD4 + T-cells and orf-virus-specific antibodies are important for the control of viral replication in the skin of infected sheep.

Immunogene therapy against mouse leukemia using B7 molecules.

B7 costimulatory molecules play an important role in T-cell activation. It is well known that tumor cells that express B7 molecules can elicit antitumor immunity, but little is known regarding which B7 molecule, B7-1 (CD80) or B7-2 (CD86), can do so more efficiently. To address this issue, we have introduced B7-1 or B7-2 into 8709 cells, a radiation-induced mouse myelocytic leukemic cell line, and have compared their potentials regarding the induction of antitumor immunity. Either B7-1- or B7-2-transduced monoclonal sublines, 8709/B7-1 or 8709/B7-2, respectively, diminished tumorigenicity in syngeneic C3H mice. Some reports have indicated that B7-1 is superior to B7-2 in the induction of antitumor immunity. Contrary to these results, the 8709/B7-2 lines are superior to the 8709/B7-1 lines in their capacity to induce antitumor immunity. In vivo depletion of lymphocyte subsets demonstrated that both CD4+ and CD8+ T cells were indispensable for B7-1- or B7-2-dependent antitumor immunity, whereas natural killer cells were not. These results suggest that in some circumstances, B7-2 molecule is more effective than B7-1 molecule in eliciting antitumor immunity.

B7-1 (CD80)-gene transfer combined with interleukin-12 administration elicits protective and therapeutic immunity against mouse hepatocellular carcinoma.

Human hepatocellular carcinoma (HCC) frequently recurs after primary therapy, resulting in poor prognosis. To try to find a way to prevent this, we examined the combined effectiveness of B7-1 (CD80)-gene transfer and interleukin-12 (IL-12) on the induction of protective antitumor immunity against poorly immunogenic BNL1ME A.7R. 1 (BNL) mouse HCC cells. We introduced mouse B7-1 gene into BNL1ME A. 7R.1 cells. Overexpression of B7-1 on BNL1ME A.7R.1 cells resulted in significant inhibititon of subcutaneous tumor development in syngeneic BALB/c mice, but not in complete rejection, suggesting that strong expression of B7-1 molecules may enhance the immunogenicity of BNL1ME A.7R.1 cells in immunocompetent mice. Lymphocyte study revealed that the cytolytic activity generated by immunization with B7-1 transfectants against BNL1ME A.7R.1 cells was mediated mainly by CD8(+) cytotoxic T lymphocytes (CTL). We examined the synergistic effect of IL-12 and immunization with B7-1 transfectants. The combination led to rejection of BNL1ME A.7R.1 cells in 6 of 10 tested mice and delayed tumor development in the remaining mice. Furthermore, the combined treatment against pre-established BNL1ME A.7R.1 tumors resulted in rejection in 3 of 8 tested mice or in significant inhibition of tumor growth in the remaining mice. In vivo lymphocyte subset depletion study indicated that the combined antitumor effect was dependent on the presence of both CD8(+) and CD4(+) T cells. In conclusion, the combination of immunization of B7-1-transfected HCC cells and IL-12 could induce protective and therapeutic immunity against parental HCC cells, and this combination may be therapeutically useful for suppressing recurrence of HCC.

Local Tumor Irradiation Augments the Response to IL-2 Therapy in a Murine Renal Adenocarcinoma

We have previously demonstrated that local tumor irradiation effectively enhanced the therapeutic effect of IL-2 therapy on pulmonary metastases from a murine renal adenocarcinoma, Renca. Irradiation with 300 rad to the left lung only, followed by systemic IL-2 therapy, results in increased tumor reduction in both lungs, suggesting that radiation enhances the systemic effect of immunotherapy. In this study, we show that irradiation of the tumor-bearing organ is essential for the combined effect of both modalities. This effect is radiation dose-dependent as increases in the radiation dosage result in greater tumor reduction in the irradiated field as well as systemically in nonirradiated fields when combined with immunotherapy. We find that irradiation has a direct inhibitory effect on Renca cell growth in vitro. Irradiation of Renca cells also causes an upregulation in H-2K d class I MHC antigen detectable at 300 rad and more pronounced with 800 rad. By in vivo selective depletion of lymphocyte subsets, we demonstrate the involvement of Lyt-2 + and L3T4 + T cell subsets and AsGM1 + cells, including NK cells, in the antitumor effect mediated by tumor irradiation and IL-2 therapy. Immunohistochemistry studies, performed on lung sections, showed a significant infiltration of CD3 + T cells and macrophages in the tumor nodules following treatment with tumor irradiation and IL-2 therapy. Our studies indicate that the mechanism of interaction between tumor irradiation and immunotherapy may include radiation-induced alterations in the tumor growth and antigenicity which may enhance or trigger an anti-tumor response elicited by IL-2 and mediated by T cells, AsGM1 + cells, and macrophages.

Reconstitution of Anti‐tumor Effects of Lentinan in Nude Mice: Roles of Delayed‐type Hypersensitivity Reaction Triggered by CD4‐positive T Cell Clone in the Infiltration of Effector Cells into Tumor

Lentinan, an antitumor polysaccharide used clinically in Japan, requires the intact T cell compartment to manifest its antitumor effects. The aim of the current study was to clarify the mechanisms playing crucial roles in the T cell requirement in the expression of antitumor effects of lentinan. Lentinan treatment of BDF1 mice transplanted intradermally with FBL‐3 induced complete tumor regression and a marked increase in survival time. The antitumor action of lentinan was abolished in mice treated simultaneously with antibodies to CD4 and CD8 antigens, whereas antibody to CD4, CD8 or NK1.1 alone was ineffective. The natural killer, cytotoxic T lymphocyte, and helper T cell activities were already augmented in this FBL‐3/BDF1 system and thus further augmentation of these activities by lentinan was not observed. These activities did not correlate with the antitumor activity of lentinan, as was confirmed in lymphocyte subset depletion experiments. On the contrary, the delayed‐type hypersensitivity (DTH) response against tumor‐associated antigens was triggered by lentinan and was abrogated only in mice treated simultaneously with antibodies to CD4 and CD8 antigens. Furthermore, a non‐cytolytic tumor‐associated antigen‐specific CD4+ T cell clone able to induce the DTH response in concert with lentinan reconstituted the antitumor effects in B6 nude mice when administered with lentinan. These results suggest that, in addition to the augmentation of immune effector cell activity against tumors, infiltration of these cells into the tumor burden initiated by the DTH responses at tumor sites may be involved in eradication of tumors by lentinan.

The Combined Use of Soybean Agglutinin and Immunomagnetic Beads for T Lymphocyte Subset Depletion of Bone Marrow Allografts: A Laboratory Analysis

The prevention of graft-versus-host disease by T-lymphocyte depletion of allografts prior to bone marrow transplantation has resulted in an increase in graft failure/rejection and relapse of disease. Evidence for the selective roles of specific T-lymphocyte subsets in each of the engraftment, graft-versus-host disease, and disease relapse processes has been presented, but sufficient clinical verification to support any hypothesis in this regard is lacking. In this paper we describe a convenient and highly flexible clinical laboratory method for depletion of specific T-lymphocytes in controllable quantities by the use of select monoclonal antibodies. Almost 3 log10 removal of CD2+ and CD8+ cells without significant loss of hematopoietic progenitors (CFU-GM, BFU-E, and CD34+ cells) can be reproducibly achieved. This method, employing soybean agglutination and immunomagnetic beads, is potentially adaptable to depletion of any cell subset or any tumor cell for which unique cell-surface antigen characteristics have been defined. In addition, our protocol is equally suited to the positive selection of stem cells and hematopoietic progenitors bearing the CD34 surface antigen.

CD4+ T-Lymphocytes prevent reactivation of toxoplasmic encephalitis (TE) in the SCID mouse

TE is common life-threatening opportunistic infection in patients with AIDS. We have recently developed a model of TE reactivation using the SCID mouse (Clin Res 1992:40;I75A). The objective of the current study was to use this model to determine the mechanisms of resistance to reactivation of TE. Immune Balb/c spleen cells were treated with monoclonal antibodies (MAb) and complement in vitro to deplete specific lymphocyte subsets prior to transfer lo SCID mice chronically infected with T.gondii. Mice then received MAb in vivo to maintain lymphocyte subset depletion. Cells depleted of both CD4+ and CD8+ cells resulted in a significant increase in mortality over controls (p = 001). Severe necrotising encephalitis associated with many T.gondii organisms were seen in CD4+/CD8+ depleted mice whereas control animals only had mild meningeal and perivascular inflammation. CD4+ or CD8+ depleted animals did not die, however whereas CD4+ depleted animals developed necrotic brain lesions associated with T.gondii., CD8+ depleted micc only developed mild meningeal and perivascular inflammation which were similar to lesions seen in controls. Depletion of CD4+ cells resulted in markedly reduced serum levels of interferon-γ (IFN-γ) and interleukin-6 (IL-6) as well as markedly impaired production of IFN-γ, IL-6, IL-4 and IL-10 (by ELISA) in splenic cultures stimulated with concanavalin A or toxoplasma lysale antigen. We conclude that in this model of TE: (I) Resistance from reactivation of TE is mediated by lymphocytes. (2) CD4+ cells appear to be more important than CD8+ cells in mediating such resistance. (3) Impairment of cytokine production due to CD4 depletion appears to play a role in reactivation of TE. (4) These results should assist in our understanding the Immunopathogenesis of TE in AIDS patients. TE is common life-threatening opportunistic infection in patients with AIDS. We have recently developed a model of TE reactivation using the SCID mouse (Clin Res 1992:40;I75A). The objective of the current study was to use this model to determine the mechanisms of resistance to reactivation of TE. Immune Balb/c spleen cells were treated with monoclonal antibodies (MAb) and complement in vitro to deplete specific lymphocyte subsets prior to transfer lo SCID mice chronically infected with T.gondii. Mice then received MAb in vivo to maintain lymphocyte subset depletion. Cells depleted of both CD4+ and CD8+ cells resulted in a significant increase in mortality over controls (p = 001). Severe necrotising encephalitis associated with many T.gondii organisms were seen in CD4+/CD8+ depleted mice whereas control animals only had mild meningeal and perivascular inflammation. CD4+ or CD8+ depleted animals did not die, however whereas CD4+ depleted animals developed necrotic brain lesions associated with T.gondii., CD8+ depleted micc only developed mild meningeal and perivascular inflammation which were similar to lesions seen in controls. Depletion of CD4+ cells resulted in markedly reduced serum levels of interferon-γ (IFN-γ) and interleukin-6 (IL-6) as well as markedly impaired production of IFN-γ, IL-6, IL-4 and IL-10 (by ELISA) in splenic cultures stimulated with concanavalin A or toxoplasma lysale antigen. We conclude that in this model of TE: (I) Resistance from reactivation of TE is mediated by lymphocytes. (2) CD4+ cells appear to be more important than CD8+ cells in mediating such resistance. (3) Impairment of cytokine production due to CD4 depletion appears to play a role in reactivation of TE. (4) These results should assist in our understanding the Immunopathogenesis of TE in AIDS patients.

Effects of in vivo T lymphocyte subset depletion on mycobacterial infections in mice.

The relative importance of CD4+ and CD8+ T cell subsets in the expression of acquired resistance to systemic infection by Mycobacterium kansasii was determined. T cell subsets were depleted in thymectomized C57BL/6 mice by the intravenous administration of monoclonal antibodies directed against the relevant T cell determinants. Depletion of the CD4+ subset exacerbated the severity of the infection in intravenously challenged mice. This effect was apparent in the first 2 weeks of the infection and persisted throughout the 12 weeks of the study. On the other hand, depletion of the CD8+ cells had no apparent effect on the growth curves. Infections by Mycobacterium tuberculosis Erdman or bacille Calmette-Guérin (BCG) Pasteur were also substantially enhanced by CD4 depletion, but not by the depletion of CD8+ cells. The effect of subset depletion on infections by M. tuberculosis and BCG was examined in both innately susceptible C57BL/6 mice and innately resistant B6D2 mice.