Modulation of T-cell functions: I. Effect of 2-mercaptoethanol and macrophages on T-cell proliferation

The in vitro effects of 2-mercaptoethanol (2-ME), macrophages (MØ), and concanavalin A (Con A) on the proliferation of normal spleen cells (NSC), MØ-depleted spleen cells (DSC), T cells, T-cell subpopulations, and B cells were assessed by [³H]thymidine incorporation. 2-ME alone was consistently shown not to be mitogenic for purified T cells; however, 2-ME enhanced the early (Days 1 and 2) Con A (2 μg/ml)-induced response of NSC, DSC, and T-cell preparations, but depressed the late response (Days 4 and 5). 2-ME alone was mitogenic for purified B-cells, as reported previously; and the 2-ME-induced B-cell response was inhibited by Con A. Preincubation of T cells with 2-ME was sufficient for enhanced Con A responsiveness; however, if 2-ME was added 24 hr after the initiation of culture, no alteration of the Con A-induced response was observed. Ly-2,3⁺ T cells were unresponsive to Con A (0.3–20 μg/ml), but the addition of 2-ME or peritoneal cells enhanced the Con A responsiveness of Ly-2,3⁺ T cells over 200-fold. Ly-1⁺ T cells responded with a similar doseresponse and kinetic profile as unselected T cells. Although Ly-1⁺ T cells responded to Con A, unlike Ly-2, 3⁺ T cells, extensive removal of MØ significantly reduced the Con A-induced responsiveness of the Ly-1⁺ T cells. The reactivities of Ly-1⁺ and Ly-2,3⁺ DSC could be reconstituted by the addition of MØ or 2-ME; however, the kinetic response of Ly-1⁺ T cells peaked on Day 2–3, and Ly-2,3⁺ T cells had a delayed response which peaked on Day 4–5. The results indicated that (i) 2-ME and/or MØ accelerate the response kinetics of T-cells to Con A; (ii) T-cell subpopulations have differential requirements for MØ and/or 2-ME in the response to Con A; (iii) T-cell subpopulations exhibit differential dose responsiveness to Con A; and (iiii) 2-ME alters Con A responsiveness by a direct effect on T cells.     

Lymphocyte Activation Gene 3 (LAG-3) Modulates the Ability of CD4 T-cells to Be Suppressed In Vivo

Lymphocyte Activation Gene – 3 (LAG-3) is an immune checkpoint molecule that regulates both T-cell activation and homeostasis. However, the molecular mechanisms underlying LAG-3’s function are generally unknown. Using a model in which LAG-3 blockade or absence reliably augmented homeostatic proliferation in vivo, we found that IL-2 and STAT5 are critical for LAG-3 function. Similarly, LAG-3 blockade was ineffective in the absence of regulatory T-cells (Treg), suggesting an important role for LAG-3 in either the responsiveness of conventional T-cells (Tconv) to regulation, or a relative defect in the ability of LAG-3 KO regulatory T-cells (Treg) to suppress the proliferation of Tconv. In this model, LAG-3 KO Treg suppressed proliferation in a manner fairly similar to wild-type (WT) Treg, but LAG-3 KO Tconv were relatively resistant to suppression. Further studies also identified a role for LAG-3 in the induction/expansion of Treg. Finally, we found that LAG-3 blockade (or knockout) led to a relative skewing of naïve CD4 T-cells toward a T_H1 phenotype both in vitro and in in vivo. Together, these data suggest that LAG-3 expression on Tconv cells makes them more susceptible to Treg based suppression, and also regulates the development of a T_H1 T-cell response.     

Regulatory function of T lymphocytes in the immune response to polyvinyl pyrrolidone. II. Characterization of the intrinsic suppressor and the induced suppressor.

Anti-PVP antibody² response is regulated by two categories of suppressor cells, the intrinsic suppressor and the induced suppressor. The intrinsic suppressor activity was dependent on the thymus-derived cells, which were relatively short-lived, insensitive to the treatment with a small dose of ATS (T₁), resistant to HC, and may be adherent to NW. On the other hand, precursor of the induced suppressor was long-lived, susceptible to ATS (T₂), and insensitive to HC. Induced suppressor itself was sensitive to HC, radiosensitive, and nonadherent to NW.These results suggest that the intrinsic suppressor and the antigen-induced suppressor belong to different subsets of T cells.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

Murine T-cell preparations: Radiosensitivity of helper activity

Sheep red blood cell (SRBC)-primed T-cells obtained by two different methods had differential helper activity kinetics and sensitivities to radiation. The helper activities of primed T-cells (pT) isolated from SRBC-immunized mice by glass wool-nylon fiber column purification and SRBC-activated T-cells (ATC) prepared by injection of thymocytes and SRBC into lethally irradiated mice were assessed in Day 5 in vitro primary immune responses to SRBC, TNP-SRBC, or TNP-KLH. The pT had more helper activity than the ATC, but pT activity was not manifested as long as ATC activity. The helper activity of 2 × 10⁶ pT was radiosensitive over an extended period after SRBC-activation; therefore, pT radiosensitivity was not temporally related to the time of activation. The helper activities of ATC and 4 × 10⁶ pT were relatively radioresistant, and 500 R enhanced ATC activity. Removal of macrophages from the ATC did not substantially effect their activities before or after irradiation. Addition of macrophages to the pT before or after irradiation enhanced their activity but not back to the level of non-irradiated (O R) pT. The helper activities of the pT and ATC resided in an anti-Thy-1 sensitive population. In fact, the 1000 R-radioresistant ATC were more sensitive to anti-Thy-1 plus complement treatment than non-irradiated ATC. The pT appeared to contain T₁ and T₂ cells, but the ATC possessed the activities mainly of T₁ cells. The evidence suggests that specific help which was the major type provided by pT cells (2 × 10⁶) was radiosensitive, but nonspecific help as induced by Con A and provided by the majority of the ATC was radioresistant.     

Early Skewed Distribution of Total and HIV-Specific CD8+ T-Cell Memory Phenotypes during Primary HIV Infection Is Related to Reduced Antiviral Activity and Faster Disease Progression

The important role of the CD8⁺ T-cells on HIV control is well established. However, correlates of immune protection remain elusive. Although the importance of CD8⁺ T-cell specificity and functionality in virus control has been underscored, further unraveling the link between CD8⁺ T-cell differentiation and viral control is needed. Here, an immunophenotypic analysis (in terms of memory markers and Programmed cell death 1 (PD-1) expression) of the CD8⁺ T-cell subset found in primary HIV infection (PHI) was performed. The aim was to seek for associations with functional properties of the CD8⁺ T-cell subsets, viral control and subsequent disease progression. Also, results were compared with samples from Chronics and Elite Controllers. It was found that normal maturation of total and HIV-specific CD8⁺ T-cells into memory subsets is skewed in PHI, but not at the dramatic level observed in Chronics. Within the HIV-specific compartment, this alteration was evidenced by an accumulation of effector memory CD8⁺ T (T_EM) cells over fully differentiated terminal effector CD8⁺ T (T_TE) cells. Furthermore, higher proportions of total and HIV-specific CD8⁺ T_EM cells and higher HIV-specific T_EM/(T_EM+T_TE) ratio correlated with markers of faster progression. Analysis of PD-1 expression on total and HIV-specific CD8⁺ T-cells from PHI subjects revealed not only an association with disease progression but also with skewed memory CD8⁺ T-cell differentiation. Most notably, significant direct correlations were obtained between the functional capacity of CD8⁺ T-cells to inhibit viral replication in vitro with higher proportions of fully-differentiated HIV-specific CD8⁺ T_TE cells, both at baseline and at 12 months post-infection. Thus, a relationship between preservation of CD8⁺ T-cell differentiation pathway and cell functionality was established. This report presents evidence concerning the link among CD8⁺ T-cell function, phenotype and virus control, hence supporting the instauration of early interventions to prevent irreversible immune damage.     

Mycobacteria activate γδ T-cell anti-tumour responses via cytokines from type 1 myeloid dendritic cells: a mechanism of action for cancer immunotherapy

Attenuated and heat-killed mycobacteria display demonstrable activity against cancer in the clinic; however, the induced immune response is poorly characterised and potential biomarkers of response ill-defined. We investigated whether three mycobacterial preparations currently used in the clinic (BCG and heat-killed Mycobacterium vaccae and Mycobacterium obuense) can stimulate anti-tumour effector responses in human γδ T-cells. γδ T-cell responses were characterised by measuring cytokine production, expression of granzyme B and cytotoxicity against tumour target cells. Results show that γδ T-cells are activated by these mycobacterial preparations, as indicated by upregulation of activation marker expression and proliferation. Activated γδ T-cells display enhanced effector responses, as shown by upregulated granzyme B expression, production of the T_H1 cytokines IFN-γ and TNF-α, and enhanced degranulation in response to susceptible and zoledronic acid-treated resistant tumour cells. Moreover, γδ T-cell activation is induced by IL-12, IL-1β and TNF-α from circulating type 1 myeloid dendritic cells (DCs), but not from type 2 myeloid DCs or plasmacytoid DCs. Taken together, we show that BCG, M. vaccae and M. obuense induce γδ T-cell anti-tumour effector responses indirectly via a specific subset of circulating DCs and suggest a mechanism for the potential immunotherapeutic effects of BCG, M. vaccae and M. obuense in cancer.     

Plasticity within the αβ+CD4+ T-cell lineage: when,how and what for?

Following thymic output, αβ⁺CD4⁺ T cells become activated in the periphery when they encounter peptide–major histocompatibility complex. A combination of cytokine and co-stimulatory signals instructs the differentiation of T cells into various lineages and subsequent expansion and contraction during an appropriate and protective immune response. Our understanding of the events leading to T-cell lineage commitment has been dominated by a single fate model describing the commitment of T cells to one of several helper (T_H), follicular helper (T_FH) or regulatory (T_REG) phenotypes. Although a single lineage-committed and dedicated T cell may best execute a single function, the view of a single fate for T cells has recently been challenged. A relatively new paradigm in αβ⁺CD4⁺ T-cell biology indicates that T cells are much more flexible than previously appreciated, with the ability to change between helper phenotypes, between helper and follicular helper, or, most extremely, between helper and regulatory functions. In this review, we comprehensively summarize the recent literature identifying when T_H or T_REG cell plasticity occurs, provide potential mechanisms of plasticity and ask if T-cell plasticity is beneficial or detrimental to immunity.     

T1 and T2 lymphocytes in primary and secondary delayed type hypersensitivity of mice. I. Contribution in the response to sheep red blood cells and to allogeneic spleen cells.

Peripheral T lymphocytes can be subdivided into two populations (T₁ and T₂ cells) based upon the short life span of T₁ cells after adult thymectomy (ATx) and sensitivity of T₂ cells to treatment with anti-thymocyte serum (ATS) in vivo. The contribution of the T₁ and T₂ cells to primary and secondary delayed type hypersensitivity (DTH) to sheep red blood cells (SRBC) and to primary DTH to allogeneic cells was studied in mice. T₂ cells were found to account for the development of the state of primary DTH responsiveness after intravenous immunization with SRBC and after subcutaneous immunization with allogeneic cells. No clear cut evidence was found that in the presence of T₂ cells DTH related T effector cells were generated from T₁ cells. In mice selectively depleted for T₁ cells by ATx, the remaining T₂ cells were capable to generate SRBC-specific T memory cells, but not in numbers as large as in non-thymectomized mice. On the other hand, T₁ cells in mice depleted for T₂ cells by ATS treatment, could give rise to normal numbers of SRBC-specific T memory cells. Apparently T₁ cells can compensate for the absence of T₂ cells during generation of T memory cells, but T₂ cells cannot do so for the loss of T₁ cells. From the time curve showing the ATx-induced decline of the population of SRBC-specific T₂ cells, involved in primary DTH responsiveness, the half life was calculated to be 6 to 7 months.     

Tregs Modulate Lymphocyte Proliferation,Activation, and Resident-Memory T-Cell Accumulation within the Brain during MCMV Infection

Accumulation and retention of regulatory T-cells (Tregs) has been reported within post viral-encephalitic brains, however, the full extent to which these cells modulate neuroinflammation is yet to be elucidated. Here, we used Foxp3-DTR (diphtheria toxin receptor) knock-in transgenic mice, which upon administration of low dose diphtheria toxin (DTx) results in specific deletion of Tregs. We investigated the proliferation status of various immune cell subtypes within inflamed central nervous system (CNS) tissue. Depletion of Tregs resulted in increased proliferation of both CD8⁺ and CD4⁺ T-cell subsets within the brain at 14 d post infection (dpi) when compared to Treg-sufficient animals. At 30 dpi, while proliferation of CD8⁺ T-cells was controlled within brains of both Treg-depleted and undepleted mice, proliferation of CD4⁺ T-cells remained significantly enhanced with DTx-treatment. Previous studies have demonstrated that Treg numbers within the brain rebound following DTx treatment to even higher numbers than in untreated animals. Despite this rebound, CD8⁺ and CD4⁺ T-cells proliferated at a higher rate when compared to that of Treg-sufficient mice, thus maintaining sustained neuroinflammation. Furthermore, at 30 dpi we found the majority of CD8⁺ T-cells were CD127^hi KLRG1^- indicating that the cells were long lived memory precursor cells. These cells showed marked elevation of CD103 expression, a marker of tissue resident-memory T-cells (T_RM) in the CNS, in untreated animals when compared to DTx-treated animals suggesting that generation of T_RM is impaired upon Treg depletion. Moreover, the effector function of T_RM as indicated by granzyme B production in response to peptide re-stimulation was found to be more potent in Treg-sufficient animals. Taken together, our findings demonstrate that Tregs limit neuroinflammatory responses to viral infection by controlling cell proliferation and may direct a larger proportion of lymphocytes within the brain to be maintained as T_RM cells.     

Colony-forming B cells in F1 hybrid and transplanted CBA/N mice.

The conditions for evaluation of suppressor cell regulation of the pokeweed mitogen (PWM)-induced plaque-forming cell (PFC) responses of peripheral blood (PB) B cells in normal individuals using allogeneic cocultures is described. In 14 separate experiments, after preincubation with concanavalin A (Con A) for 2 days, PB cells suppressed the PWM-induced anti-sheep erythrocyte (SRBC) PFC response of fresh allogeneic PB cells to 17% of the expected PFC response (P < 0.05). In addition, control cells incubated for 2 days in the absence of Con A suppressed the PWM- induced PFC response of allogeneic cells in 6 of 14 experiments to the same extent as did the Con A-generated cells (P < 0.01). It was found that unstimulated control cells (without Con A activation) from normal subjects who themselves were nonresponders to PWM stimulation (< 50 PFC/10⁶ cells) usually suppressed the PFC response of allogeneic cells (P < 0.05), while control cells from normal subjects who consistently had a good PFC response to PWM stimulation (> 75 PFC/10⁶ cells) did not suppress the PFC response of allogeneic cells. The spontaneously occurring suppressor cell in nonresponder PB cell suspensions was sensitive to 3000-R irradiation, and the nonresponder state was not associated with a decreased blastogenic response to PWM. Thus, some normal subjects who themselves had a poor PWM-induced PFC response had irradiation-sensitive, spontaneously occurring suppressor cells which were capable of suppressing the PWM-induced PFC response of normal responders. The majority of normal subjects (90%) were good PFC responders to PWM stimulation and did not spontaneously suppress the PFC response of allogeneic cells to PWM, but did have PB cells which were capable of being activated by Con A to suppress.     

MHC recognition by clones of Mls specific T-lymphocytes

To test whether M1s determinants, like other non-MHC or nominal antigens, are recognized by T-cells in association with H-2 determinants, the in vitro proliferative responses of T-cell lines and clones were studied. Lines and clones were prepared by soft agar cloning (B10.BR x BALB/c)F₁ (H-2^k/H-2^d, M1s^b/M1s^b) T-cells responding in a primary MLR to AKD2F1 (H-2^k/H-2^d, M1s^a/M1s^a) stimulator cells. All the T-cell clones obtained could respond equally well in a proliferative assay to the Mls^a determinant in association with the H-2 haplotype of either parent, i. e., DBA/2 (H-2^d, M1s^a), and AKR (H-2^k, M1s^a) both stimulated equally well. When the T-cell lines and clones were screened against stimulators from recombinant inbred (RI) strains, it became apparent that strains exhibiting the H-2^b, M1s^a genotype stimulated poorly or not at all. This shows that the T-cell response to M1s^a involves MHC recognition, and raises the possibility that the response to M1s^a can involve recognition of H-2 specificities shared between the H-2 ^k and H-2 ^d haplotypes.Abbreviations used in this paper MHC major histocompatibility complex - MLC mixed lymphocyte culture - IL-2 interleukin 2 - Con A concanavalin A - RI recombinant inbred Howard Hughes Medical Institute     

Immune interferon induction by T-cell mitogens involves different T-cell subpopulations.

Recent studies of mitogen-induced DNA synthesis in cells from various lymphoid tissues indicate that certain mitogens may selectively activate specific T-cell subpopulations. Staphylococcal enterotoxin A (SEA) optimally stimulates thymocytes and spleen cells (presumed T1 cells) and phytohemagglutinin P (PHA) optimally stimulates lymph node and spleen cells (presumed T2 cells); concanavalin A (Con A) stimulates cells from all these sources well. To determine further the specificity of mitogens for T-cell subpopulations, immune interferon (IIF) production was studied in spleen cells from mice treated in vivo with cortisone acetate (CA), preferentially a T1-cell inactivator, and antithymocyte serum (ATS), preferentially a T2-cell inactivator. The effect of administering gallic acid (3,4,5 trihydroxy-benzoic acid) (GA) in vivo was also studied, since in vitro studies showed GA to be capable of blocking IIF production. Results indicate that SEA induces IIF primarily in T1 cells, PHA induces IIF primarily in T2 cells, and Con A induces IIF in both T1 and T2 cells. Furthermore, GA was shown to mimic the ability of CA to inactivate T1 cells selectively in vivo. The data indicate that more than one type of T-cell subpopulation is capable of producing IIF.     

Toll-like receptor (TLR) expression on CD4 and CD8 T-cells in patients chronically infected with hepatitis C virus

Toll-like receptor (TLR) expression on T-cells and the signalling pathways that lead to the production of cytokines may limit antigen-specific T-cell responses. Here, expression of TLR and retinoic acid inducible gene I (RIG-I) on T-cells were evaluated in patients chronically infected with hepatitis C virus (HCV), before and during pegylated interferon-α and ribavirin therapy. Expression of TLR2,3,4,7,9 and retinoic acid inducible gene (RIG)-I on different CD4⁺ and CD8⁺ T-cell sub-populations (naïve: CD45RA⁺CD57⁻; central memory: T_CM CD45RA⁻CD57⁻; effector memory: T_EM CD45RA⁻CD57⁺ and terminally differentiated effector memory: T_EMRA CD45RA⁺CD57⁺) were measured by flow cytometry. TLR7, TLR9 and RIG-I expression on CD4⁺ T-cells and RIG-I expression on CD8⁺ T-cells was higher in patients than healthy controls. Therapy increased expression of TLR2, TLR4 and TLR9 and this was observed for all T-cell sub-populations. Evaluation of TLR expression at baseline did not identify patients able to achieve sustained virological response following therapy.     

Quantitation of the T cell deficiency in RFM mice with host versus graft disease.

Host versus graft (HVG) syndrome is the fatal complex of lesions which has been observed in six inbred strains of mice following the perinatal inoculation of related F₁ hybrid spleen cells. Morphological studies have indicated that the key lesion is the depletion of peripheral T lymphocytes due to inflammatory destruction and failure of the thymus to replace them. In the present studies, tests of T-cell function were done on RFM mice, which had developed HVG disease following perinatal inoculations of (T₆ × RFM)F₁ spleen cells. As compared to control values, HVG spleen cell suspensions showed loss of reactivity to phytohemagglutinin (PHA) = 90%, to concanavalin A (Con A) = 94%, to (T₆ × RFM)F, cells in the mixed lymphocyte reaction (MLR) = 82%, to DBA cells in MLR = 94%, and to DBA mastocytoma cells in cell-mediated lympholysis (CML) = 95%. Lymph node cell suspensions showed losses of reactivity to PHA = 83%, to Con A = 62%, to (T₆ × RFM)F₁ cells in the MLR = 91%, and to DBA cells in the MLR = 77%. The CML activity of nodal cells to DBA mastocytoma cells varied widely from 12 to 273% of control values, and averaged 121%. Filtration of HVG spleen cells through nylon fiber columns failed to restore low responses to PHA to normal values. This suggested that the macrophage-like, adherent accessory cells were not acting as suppressors of T-cell responses in HVG disease. The deficits in all T-cell-mediated functions tested so far, appeared to correlate very well with quantitative morphological studies which showed the loss of 98% of the small lymphocytes normally present in the thymic dependent portions of the splenic white pulp. It is suggested that experimental HVG disease may serve as a model for immunodeficiency syndromes of the Nezelof type which are also characterized by T-cell deficiency, poor primary antibody responses, and the presence of variable amounts of serum immunoglobulins.     

In vitro inhibition of the helper activity of GAT-specific T-cell lines by a syngeneic anti-idiotypic serum: preferential effect on the IgG response

We described in this paper the characteristics of a syngeneic anti-idiotypic serum made in BALB/c against BALB/c anti-poly (Glu⁶⁰ Ala³⁰ Tyr¹⁰) (GAT) antibodies. This serum recognizes idiotypic determinants present in all anti-GAT sera whatever the allotypic markers of the mice used to prepare the sera. The functional effect of this serum on two helper cell lines is also described. Cell line BDF₁/52 was obtained from GAT immunized lymph node cells (LNC). Cell line BDF₁/E₃ was selected from splenic T-cells educated in vitro on GAT-pulsed adherent cells. Both lines were propagated in presence of filler cells, antigen, and medium containing T-cell growth factor(s) from splenic cells activated with concanavalin A. Both cell lines exhibit a helper activity as measured by the plaque-forming cell (PFC) response they induce in vitro in the presence of DNP-GAT and DNP sensitized B cells. Their helper activity is specific and they require a hapten-carrier bridge to activate B cells. These lines are able to induce IgG₁, IgG_2a and IgG_2b anti-TNP PFC. Syngeneic anti-idiotypic serum B 658 inhibits specifically the function of these two lines but does not affect the helper activity of an OVA-specific T-cell line. The blocking activity of the serum can be adsorbed on a hybridoma protein with anti-GAT activity. This inhibition affects more dramatically the IgG₁ response than the IgG_2a and IgG_2b responses.     

Soluble suppressor factor produced by pokeweek mitogen-stimulated human peripheral blood leukocytes

Human peripheral blood leukocytes were exposed to either PWM or Con A mitogens. Cells activated by both these mitogens were able to depress proliferation in an MLC, and to inhibit the generation of spontaneous killer cell (SK) and induced T-cell cytotoxic activity. PWM-activated cells incubated in media for 48 hr were able to elaborate a soluble factor in vitro. This factor suppressed cytotoxicity, and was active only when present at the initiation of MLC cultures. In contrast, cells exposed to Con A were able to suppress immune responsiveness, but this population did not release a soluble factor which could inhibit cytotoxicity. PWM induction appears to be dependent on phagocytic cells, while Con A activation is less dependent on this adherent population. An enriched adherent cell population, stimulated with PWM, was able to suppress cytotoxicity. Thus, the PWM-stimulated system of suppression is mediated through a soluble factor and is dependent on adherent cells.     

Human Vδ1 γδ T cells expanded from peripheral blood exhibit specific cytotoxicity against B-cell chronic lymphocytic leukemia-derived cells

Background aimsThere is increasing interest in using γδ T cells (GDTC) for cancer immunotherapy. Most studies have been concerned with the Vδ2 subset in blood, for which several expansion protocols exist. We have developed a protocol to expand Vδ1 and Vδ2 preferentially from human blood. We have characterized these subsets and their specificities for leukemic targets.MethodsGDTC were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors via positive magnetic cell sorting; their proliferation in vitro was induced by exposure to the mitogen concanavalin A (Con A). CD107 and cytotoxicity (Cr⁵¹-release and flow cytometric) assays were performed. GDTC clones and target cells were immunophenotyped via flow cytometry.ResultsLonger initial exposure to Con A typically resulted in higher Vδ1 prevalence. Vδ1 were activated by and cytotoxic to B-cell chronic lymphocytic leukemia (B-CLL)-derived MEC1 cells, whereas Vδ2 also responded to MEC1 but more so to the Philadelphia chromosome-positive [Ph+] leukemia cell line EM-enhanced green fluorescent protein (2eGFPluc). Vδ2 clone cytotoxicity against EM-2eGFPluc correlated with Vδ2 T-cell antigen receptor (TCR) and receptor found on Natural Killer cells and many T-cells (NKG2D), whereas Vδ1 clone cytotoxicity versus MEC1 correlated with Vδ1 TCR, CD56 and CD95 expression. Vδ1 also killed Epstein-Barr Virus (EBV)-negative B-CLL-derived TMD2 cells. Immunophenotyping revealed reduced HLA-ABC expression on EM-2eGFPluc, whereas MEC1 and TMD2 exhibited higher Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAILR1).ConclusionsOur ability to expand peripheral Vδ1 cells and show their cytotoxicity to B-CLL-derived cell lines suggests that this novel approach to the cellular treatment of B-CLL may be feasible.     

Direct evidence for the response of B and T cells to pokeweed mitogen

Chicken spleen cells containing chromosomally marked thymus derived (T) and bursa-derived (B₂) cells were evaluated for their ability to respond to pokeweed mitogen (PWM), concanavalin A (Con A), and to anti-immunoglobulin serum during a 4-day culture period. The results indicate that soluble PWM induces a proliferative response of B₂ cells in addition to a predominant T cell response. The PWM-induced B₂ cell proliferative response was clearly detected only at 4 days after culture initiation. Soluble Con A did not induce detectable proliferation of B₂ cells and stimulated T cells exclusively. In contrast, anti-immunoglobulin serum was a specific stimulant for B₂ cells under the culture conditions used.