scholarly journals GENETIC CONTROL OF THE IMMUNE RESPONSE

1974 ◽  
Vol 140 (4) ◽  
pp. 977-994 ◽  
Author(s):  
Peter Lonai ◽  
Hugh O. McDevitt
Keyword(s):  
T Cells ◽  
B Cell ◽  
Tritiated Thymidine ◽  
Lymphocyte Transformation ◽  
Cross Reactivity ◽  
Pokeweed Mitogen ◽  
Thymidine Uptake ◽  
Soluble Antigen

In vitro antigen-induced tritiated thymidine uptake has been used to study the response of sensitized lymphocytes to (T,G)-A--L, (H,G)-A--L, and (Phe,G)-A--L in responder and nonresponder strains of mice. The reaction is T-cell and macrophage dependent. Highly purified T cells (91% Thy 1.2 positive) are also responsive, suggesting that this in vitro lymphocyte transformation system is not B-cell dependent. Lymphocytes from high and low responder mice stimulated in vitro react as responders and nonresponders in a pattern identical to that seen with in vivo immunization. Stimulation occurs only if soluble antigen is added at physiological temperatures; antigen exposure at 4°C followed by washing and incubation at 37°C fails to induce lymphocyte transformation. Stimulation is specific for the immunizing antigen and does not exhibit the serologic cross-reactivity which is characteristic of these three antigens and their respective antisera. The reaction can be inhibited by anti-H-2 sera but not by anti-immunoglobulin sera. The anti-immunoglobulin sera did, however, inhibit lipopolysaccharide or pokeweed mitogen stimulation. These results suggest that the Ir-1A gene(s) are expressed in T cells, and that there are fundamental physiologic differences between T- and B-cell antigen recognition.

A CD19/CD3 Bispecific Tandab, AFM11, Recruits T Cells To Potently and Safely Kill CD19+ Tumor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4405-4405
Author(s):  
Eugene Zhukovsky ◽  
Uwe Reusch ◽  
Carmen Burkhardt ◽  
Stefan Knackmuss ◽  
Ivica Fucek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cancer Cells ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Cross Reactivity ◽  
Equity Ownership

Abstract To harness the potent tumor-killing capacity of T cells for the treatment of CD19+ malignancies, we developed a humanized bispecific tetravalent antibody, with two binding sites for CD3 and CD19, the CD19/CD3 RECRUIT-TandAb AFM11. CD19 is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies such as Non Hodgkin Lymphoma. Since native antibodies cannot recruit T cells, we engineered a bispecific anti-CD19/anti-CD3 TandAb. The tumor-specific CD19 antigen module targets the TandAb to cancer cells, while simultaneously, the CD3 effector module recruits and activates T cells, leading to cancer cell lysis. The advantages of the TandAb technology, relative to other bi-functional fragment antibody scaffolds, include: improved pharmacokinetics (PK) enabling intravenous dosing, more drug-like properties, and avidity-enhanced efficacy for the targeting and killing of tumor cells. We evaluated in vitro efficacy and safety using CD19+ cell lines, and in vivo efficacy in a murine NOD/scid xenograft model reconstituted with human PBMC. Further, we used standard preclinical IND enabling assays to evaluate tissue cross reactivity, PK, and toxicological profile (local tolerance, hematocompatibility, effects on hematopoesis, etc). In vitro assays demonstrated the higher potency and efficacy of target cell lysis by AFM11 relative to a bispecific tandem scFv (that is currently in clinical evaluation). CD8+ T cells dominate early AFM11-mediated cytotoxicity (4 hrs) while after 24 hrs both CD4+ and CD8+ T cells equally contribute to tumor lysis with EC50 between 0.5 – 5 pM; cytotoxicity was independent of CD19 cell-surface density. AFM11 exhibited similar cytotoxicity over effector:target ratios ranging from 5:1 to 1:5, and facilitated serial T cell-killing of its targets. The advantage of AFM11 over the bispecific tandem scFv was most pronounced at lower effector:target ratios. AFM11 activated T cells only in the presence of CD19+ cells. In PBMC cultures, AFM11 induced CD69 and CD25 expression, T cell proliferation, and production of IFN-γ, TNF-α, IL-2, IL-6, and IL-10. Depletion of CD19+ cells from PBMC abrogated these effects, demonstrating that the T cell activation is strictly CD19+ target-dependent. Thus, AFM11 should not elicit the devastating cytokine release observed when full-length antibodies bind CD3. Up to one week co-incubation with AFM11 did not inhibit T cell cytotoxicity, suggesting that the TandAb does not induce anergy. In vivo, AFM11 induced dose-dependent growth inhibition of Raji tumors; a single 0.5 mg/kg dose exhibited efficacy similar to 5 daily injections. In the tissue cross reactivity study, only tissues containing CD19+ and CD3+ cells were stained by AFM11; all other tissues, including vital organs, displayed no cross reactivity. Similarly, no local intolerance was observed in rabbits, and no effect on myeloid and erythroid progenitors was observed in a colony-forming assay. Strong accumulation of 125I-labeled AFM11 was observed in the tumors of mice engrafted with CD19+ cancer cells, and no unspecific organ accumulation was observed. Finally, evaluated on the basis of Cmax and the area under the curve (AUC), AFM11 exhibited dose linearity (20 – 500 mg AFM11 dose range) upon single i.v. bolus administration in mice; half-life (T1/2) ranged from 18.4 to 22.9 hr. In summary, AFM11 is a highly efficacious novel drug candidate for the treatment of CD19+ malignancies with an advantageous safety profile and anticipated dosing regimen. Disclosures: Zhukovsky: Affimed Therapeutics AG: Employment, Equity Ownership. Reusch:Affimed Therapeutics AG: Employment. Burkhardt:Affimed Therapeutics AG: Employment. Knackmuss:Affimed Therapeutics AG: Employment. Fucek:Affimed Therapeutics AG: Employment. Eser:Affimed Therapeutics AG: Employment. McAleese:Affimed Therapeutics AG: Employment. Ellwanger:Affimed Therapeutics AG: Employment. Little:Affimed Therapeutics AG: Consultancy, Equity Ownership.

scholarly journals Marked Immunosuppression With Minimal Myelosuppression by Bleomycin In Vitro

Blood ◽  
1973 ◽  
Vol 41 (5) ◽  
pp. 721-726 ◽  
Author(s):  
Glenn Tisman ◽  
Victor Herbert ◽  
Le Teng Go ◽  
Lois Brenner
Keyword(s):  
T Cells ◽  
Potent Inhibitor ◽  
Lymphocyte Transformation ◽  
New Drugs ◽  
Pokeweed Mitogen ◽  
Variable Response ◽  
In Vitro System ◽  
Lymphoid Neoplasia

Abstract Bleomycin, in concentrations similar to those present after drug administration, was a potent inhibitor in vitro of both human phytohemagglutinin (PHA)-stimulated and pokeweed mitogen (PMW)-stimulated lymphocyte transformation, but lower concentrations were much more inhibitory to the former. Bleomycin showed minimal or no in vitro myelosuppression. The similarity of these in vitro results to in vivo results by others suggests the in vitro system used here may be useful to screen new drugs for potential in vivo suppression of either the myeloid or the lymphoid tissue. The data suggest that by adjusting the dose of bleomycin inhibition of lymphocytes stimulated by PHA with only minimal inhibition of lymphocytes stimulated by PWM may be possible. Variable response of lymphoid neoplasia to bleomycin may in part reflect whether the cell type is T or B in origin, in view of work by others suggesting that PHA is more stimulatory to T cells and PWM is stimulatory to both B and T cells.

scholarly journals THE EFFECT OF SKIN HOMOGRAFT REJECTION ON RECIPIENT AND DONOR MIXED LEUKOCYTE CULTURES

1965 ◽  
Vol 122 (4) ◽  
pp. 651-664 ◽  
Author(s):  
Joost J. Oppenheim ◽  
Jacqueline Whang ◽  
Emil Frei
Keyword(s):  
Mononuclear Cells ◽  
Tritiated Thymidine ◽  
White Blood Cells ◽  
Calf Serum ◽  
Lymphocyte Transformation ◽  
Humoral Factors ◽  
Unstimulated Control ◽  
Graft Donor

The lymphocyte proliferation in repeatedly studied mixed leukocyte cultures of peripheral white blood cells from a skin graft donor and 2 recipients was significantly increased at the time of graft rejection. This was determined from the increased proportions of mononuclear cells labeling with tritiated thymidine, increased mitotic indices, and the appearance of increased numbers of transformed lymphocytes after rejection of 1st and 2nd skin grafts. The temporarily enhanced response occurred sooner and was of shorter duration after the second than after the first graft, but was quantitatively similar each time. The cell proliferation in the mixed leukocyte cultures of the two recipients was similarly affected by the homograft rejections. The cultures containing three cell populations usually manifested a greater lymphocyte response than corresponding cultures of leukocytes from only two unrelated subjects. An increase in the ratio of female recipient to male graft donor metaphases in the cultures at the time of enhanced lymphocyte transformation indicated that proliferation of the graft recipient lymphocytes was responsible for the above findings. Unmixed, unstimulated control cultures grown in autologous, the other subjects plasma, or heterologous calf serum failed to support significant lymphocyte transformation. The role of humoral factors and relationship of the in vitro cellular responses to the in vivo homograft reaction are discussed.

Platelet Membranes/Drug Combination as Antigenic Stimulus for Lymphocytes of Patients with Autoimmune Quinine- or Quinidine-Dependent Purpura

1979 ◽  
Author(s):  
P. K. Hosseinzadeh ◽  
B. G. Firkin ◽  
S. L. Pfueller
Keyword(s):  
B Cell ◽  
Drug Combination ◽  
Lymphocyte Transformation ◽  
Pokeweed Mitogen ◽  
Induced Response ◽  
Thymidine Uptake ◽  
Membrane Component ◽  
Igg Antibody ◽  
Drug Induced ◽  
Antigenic Stimulus

Quinine- or quinidine-induced thrombocytopaenia appears due to synthesis of an IgG antibody which causes platelet damage. We have studied the nature of the antigenic stimulus in this disorder by measuring the incorporation of 3H-thymidine into DNA of normal or patients’ lymphocytes cultured in the presence of the drugs in comparison to known mitogens (phytohaemagglutin-P or pokeweed mitogen).’ Patients’ lymphocytes responded normally to mitogens but not to the drugs, heterologous or autologous platelets alone. However, significant thymidine uptake occurred when patients’ lymphocytes were cultured with autologous or heterologous platelets in the presence of therapeutic concentrations of the drugs (2.4 x 10-4 . 2.4 x 10-8M). Lymphocyte transformation was maximal after 7 days (up to 93% of phytohaemagglutinin-induced response), suggesting B cell involvement. Supernatants from platelets which had undergone the release reaction could not replace platelets while membranes were more effective than platelets on-a protein basis. Control lymphocytes from 20 normals and 8 patients with non-drug-induced thrombocytopaenia did not demonstrate transformation by the drugs and platelets. Thus the drug in combination with a platelet membrane component act as an antigenic stimulus only for sensitised lymphocytes.

scholarly journals Immunologic and Cytogenetic Studies of Chronic Lymphocytic Leukemic Cells

Blood ◽  
1965 ◽  
Vol 26 (2) ◽  
pp. 121-132 ◽  
Author(s):  
JOOST J. OPPENHEIM ◽  
JACQUELINE WHANG ◽  
EMIL FREI
Keyword(s):  
Tritiated Thymidine ◽  
Lymphocyte Transformation ◽  
Normal Subjects ◽  
Peripheral Circulation ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Serum Factors ◽  
Cytogenetic Studies

Abstract The lymphocyte transformation response of 17 chronic lymphocytic leukemia patients when tested in the short-term tissue culture with PHA-M, and PPD was found to be significantly decreased when compared to normal subjects. Serum factors were not found to be responsible for this cellular hyporesponsiveness. The proportions of immunoresponsive lymphocytes found in the patients’ peripheral circulation decreased as their white blood cell count increased. The transformation response to PHA-M was generally better than to PPD. Neither the PPD negative patients nor the normal PPD negative subjects’ cells responded to PPD stimulation in vitro. Monocytes usually would phagocytize particles added to the cultures and could thus be distinguished from the nonphagocytic proliferating lymphocytes which were the only cells that incorporated thymidine H3. Radioautographs of tritiated thymidine also revealed the rate of PPD lymphocyte transformation to be slower than with PHA-M. There were no significant differences in the proportions or the degree of leukemic and normal transformed lymphocyte labeling with tritiated thymidine. Cytogenetic studies revealed that the patients’ mitotic indices both in vivo and in vitro were markedly depressed. The modal chromosome number was 46 in each patient, and no cytogenetic abnormalities other than those due to exposure to radiation were found.

‘In vivo’ and ‘in vitro’ activity of Larrea divaricata Cav. on EL-4 cells

2010 ◽  
Vol 30 (8) ◽  
pp. 965-971 ◽  
Author(s):  
Roberto Davicino ◽  
Rosario Alonso ◽  
Claudia Anesini
Keyword(s):  
Cell Proliferation ◽  
Aqueous Extract ◽  
Tritiated Thymidine ◽  
Folk Medicine ◽  
Thymidine Uptake ◽  
Stimulatory Action ◽  
T Lymphoma ◽  
Larrea Divaricata

Larrea divaricata is a plant widely used in folk medicine in Argentina. It has been demonstrated that an aqueous extract of L. divaricata possesses a biphasic effect on cell proliferation, at low concentrations exerts a stimulatory action and at high concentrations exerts anti-proliferative effects upon the T lymphoma BW 5147; therefore, we propose in this paper to test the effect of the extract ‘in vitro’ and ‘in vivo’ in another T-cell lymphoma named EL-4. It was analyzed ‘in vitro’ cell proliferation by tritiated thymidine uptake and the effect of the extract on tumors induced in mice analyzing tumor progression and survival.The results showed that the aqueous extract induced the proliferation of tumor cells at all the concentrations studied. The results ‘in vivo’ showed that the aqueous extract stimulated significantly the size of tumors and that untreated mice lived longer than those treated. It is important to be very careful when plant extracts are selected for the treatment of several diseases. Consequently, before using a plant extract, specific scientific studies must be undertaken on different models to certificate therapeutic and adverse effects. Moreover, it can be said that L. divaricata has a specific anti-tumor mechanism of action depending on the targets.

Superior Immunogenicity of Idiotype Fab Fragments As Compared to Entire Immunoglobulin for Active Lymphoma Immunotherapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1633-1633
Author(s):  
Marcelo A. Navarrete ◽  
Benjamin Kisser ◽  
Hendrik J. Veelken
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
B Cell Lymphomas ◽  
Cd8 Cells ◽  
Fab Fragments ◽  
The Individual

Abstract Abstract 1633 Introduction: The individual collection of epitopes within the variable regions of the unique immunoglobulin expressed by every mature B-cell lymphoma (idiotype, or Id) represents a tumor-specific antigen and lends itself as a target for therapeutic vaccination strategies. Immunization with tumor Id has the capacity to elicit polyclonal antibody responses as well as CD8+ and CD4+ T cells recognizing Id-derived peptides presented on class I and class II HLA molecules, respectively. Due to a perceived low immunogenicity of lymphoma-derived Id, most Id vaccines tested in clinical trials so far have been formulated as conjugates with the strongly immunogenic carrier keyhole limpet hemocyanin (KLH). In contrast, we have consistently observed high rates of humoral and cellular anti-Id immune responses in consecutive trials of active immunization with unconjugated recombinant Fab fragments of Id in indolent B-cell lymphomas (Bertinetti et al., Cancer Res. 2006; Navarrete et al., BLOOD 2011). We therefore hypothesized that Id Fab fragment might be intrinsically more immunogenic than entire Id Ig and tested this hypothesis by comparative in vitro experiments. Methods: Monocyte-derived dendritic cells (DC) where loaded with human monoclonal IgG, papain-digested Fab fragments, Fc fragments, or recombinant lymphoma-derived Fab fragments. Functional DC phenotypes were assessed by flow cytometry of crucial maturation and activation markers. IL-10 and IL-12 was measured in DC culture supernatants by ELISA. Antigen-loaded DC where subsequently used for priming of CFSE-labeled autologous peripheral blood mononuclear cells. Stimulated T cell populations were analyzed by multicolor flow cytometry. Results: Loading of DC with Fab, Fc, IgG, or mixtures of Fab and Fc fragments did not alter surface expression of CD11c, CD80, CD83, CD86, HLA-DR, PDL-1 and PDL-2 on DC. Likewise, the various antigens did not influence the cytokine release by DC during the loading or maturation process. DC loaded with isolated Fab fragments induced significantly higher proliferation of both CD4+ and CD8+ T cells than undigested IgG. The mean proliferation rate of CD4+ cells stimulated with Fab fragments was 18.5% versus 5.6% for undigested IgG stimulation (p=0.021); proliferation rates of CD8+ cells were 14.2% versus 6.2% (p=0.034). These results were reproduced for 4 different monoclonal IgGs tested on 4 different donors. The addition of Fc fragments to Fab reduced the proliferation rates of CD4+ and CD8+ cells to 10.2% and 8.6% respectively. In addition, DC loaded with undigested IgG induced a relative increase in the number of CD25high/FoxP3+ regulatory T cells compared with Fab stimulation (8.2% versus 1.4%; p<0.01). Conclusions: Isolated Fab fragments, i.e. the Id portions that contain the individual candidate antigenic epitopes of B-cell lymphomas, prime autologous T cells in vitro more efficiently than entire IgG. This finding is consistent with the high immune response rate against recombinant unconjugated Fab fragments observed in vivo in our clinical vaccination trials. Peptide sequences shared between Ig molecules that are predominantly located in the IgG Fc fragment appear to exert an inhibitory effect on T-cell priming. In accordance with our recent in vivo data in a syngeneic mouse model of Id vaccination (Warncke et al., Cancer Immunol. Immunother. 2011), this effect may be mediated by effective activation of Treg. Fab fragments therefore appear to be the more immunogenic and therefore preferable Ig antigenic format for active anti-Id immunotherapy. Furthermore, the inhibitory effects of IgG Fc offers a potential explanation for the recently reported lack of efficacy of Id vaccination in IgG-expressing follicular lymphomas in a randomized phase III trial, in which patients with IgM-expressing lymphomas, in contrast, had a significant benefit from Id vaccination in intention-to-treat analyses (Schuster et al., JCO 2011). Disclosures: No relevant conflicts of interest to declare.

scholarly journals The in vitro induction of immunological tolerance in the B lymphocyte by oligovalent thymus-dependent antigens.

1975 ◽  
Vol 141 (5) ◽  
pp. 962-973 ◽  
Author(s):  
J W Schrader
Keyword(s):  
T Cells ◽  
B Cell ◽  
Gamma Globulin ◽  
Tolerance Induction ◽  
Marginal Effect ◽  
Cell Tolerance ◽  
B Cell Tolerance ◽  
In Vitro System

B-cell tolerance has been induced by oligovalent thymus-dependent antigens in an entirely in vitro system. Dissociated spleen cells from congenitally athymic (nu/nu) mice were preincubated for 24 h with 0.1 -- 1 mg/ml of either fowl gamma globulin (FGG) of DNP-human gamma globulin (DNP-HGG). After washing, the cells were tested for the ability to mount in vitro, thymus-independent responses against FGG and DNP. A state of specific responsiveness to either FGG or DNP was thus demonstrated. Features of this wholly in vitro system that paralleled previous findings on the in vivo induction of B-cell tolerance in nu/nu mice were the kinetics, 24 h being required for tolerance induction in either case, the abrogation of tolerance induction by the presence of POL both in vivo and in vitro, and finally the observation that in neither case was there a requirement for the antigens to be deaggregated. It was shown that DNP-(Fab) 2 fragments prepared from HGG induced DNP-specific tolerance indicating that the Fc piece was not required for tolerance induction in this in vitro system. DNP-bovine serum albumin was less effective than DNP-HGG or DNP-(Fab)2. Preincubation with subtoxic concentrations of DNP-lysine of DNP-epsilon-capric acid had only a marginal effect on DNP responsiveness. Since nu/nu mice, lacking in detectable T-cell function, were used as spleen cell donors, this work provides further evidence that B-cell tolerance to thymus-dependent antigens can be induced without the participation of T cells. It is suggested that B-cell tolerance to thymus-dependent antigens occurs when the antigen in a sufficient concentration and over a sufficient period of time has direct access to the B cell. This contact with antigen must be in the absence of an additional influence provided either by adjuvants like endotoxin or POL, or by activated macrophages, which may be stimulated by activated T cells; otherwise not tolerance but B-cell activation will occur.

scholarly journals Induction of in vitro differentiation and immunoglobulin synthesis of human leukemic B lymphocytes.

1978 ◽  
Vol 148 (6) ◽  
pp. 1570-1578 ◽  
Author(s):  
S M Fu ◽  
N Chiorazzi ◽  
H G Kunkel ◽  
J P Halper ◽  
S R Harris
Keyword(s):  
T Cells ◽  
B Cells ◽  
Plasma Cells ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Pokeweed Mitogen ◽  
In Vitro Differentiation ◽  
Immunoglobulin Synthesis

Successful induction of in vitro differentiation and immunoglobulin synthesis of the leukemic lymphocytes was carried out in two cases of chronic lymphocytic leukemia. Few plasma cells and little specific Ig secretion were detected in the cultures of isolated leukemic B cells in either the presence or the absence of autologous T cells. Up to 30% of the leukemic B cells matured to plasma cells, and a 32-fold increase in specific Ig synthesis was observed when T cells from normal individuals were added to the cultures of these leukemic B cells. In one of the two cases, autologous T cells were able to induce greater than 50% of the leukemic B cells to differentiate further to plasma cells in the presence of pokeweed mitogen. This markedly accelerated in vitro differentiation was only achieved with leukemic cells from cases in which there was evidence of slight differentiation in vivo. No evidence could be obtained for excessive suppressor T cells in these patients. However, a T-cell defect in the generation of allogeneic effect helper factors was identified. This defect may be responsible for the reduced rate of leukemic maturation in vivo.

scholarly journals Participation of suppressor T cells in the immunosuppressive activity of a heteroantiserum to human Ia-like antigens (p23,30).

1980 ◽  
Vol 151 (1) ◽  
pp. 257-262 ◽  
Author(s):  
S Broder ◽  
D L Mann ◽  
T A Waldmann
Keyword(s):  
T Cells ◽  
B Cell ◽  
Inhibitory Effect ◽  
Pokeweed Mitogen ◽  
Suppressor T Cells ◽  
Immunosuppressive Activity ◽  
Immunoglobulin Production ◽  
Cells Cultured ◽  
Simple Interaction

We studied the effects of an antiserum to human Ia-like antigens (p23,30) upon the polyclonal activation of normal B cells (cultured with various combination of irradiated and unirradiated T cells) to become immunoglobulin-secreting cells after stimulation with pokeweed mitogen in vitro. We found that the antiserum suppressed immunoglobulin production. The inhibitory effect did not appear to result from a simple interaction at the B-cell/monocyte level alone. Rather, the inhibitory effect required the presence of a radiosensitive subset of autologous suppressor T cells.

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