Gammadelta T cells enhance the expression of experimental autoimmune encephalomyelitis by promoting antigen presentation and IL-12 production

Using an adoptive transfer model of experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (MBP)-reactive lymph node cells (LNC), we have shown that depletion of gammadelta T cells from LNC resulted in diminished severity of EAE in recipient mice, both clinically and histopathologically. The reduced potency of gammadelta T cell-depleted LNC to induce EAE correlated with decreased cell proliferation in response to MBP. The gammadelta T cell effect upon the threshold of MBP-induced LNC proliferation and EAE transfer was restored by reconstitution of gammadelta T cells derived from either MBP-immunized or naive mice, indicating that this effect was not Ag specific. The enhancing effect of gammadelta T cells on MBP-induced proliferation and EAE transfer required direct cell-to-cell contact with LNC. The gammadelta T cell effect upon the LNC response to MBP did not involve a change in expression of the costimulatory molecules CD28, CD40L, and CTLA-4 on TCRalphabeta(+) cells, and CD40, CD80, and CD86 on CD19(+) and CD11b(+) cells. However, depletion of gammadelta T cells resulted in significant reduction in IL-12 production by LNC. That gammadelta T cells enhanced the MBP response and severity of adoptive EAE by stimulating IL-12 production was supported by experiments showing that reconstitution of the gammadelta T cell population restored IL-12 production, and that gammadelta T cell depletion-induced effects were reversed by the addition of IL-12. These results suggest a role for gammadelta T cells in the early effector phase of the immune response in EAE.     

Adoptively transferred experimental autoimmune encephalomyelitis in SJL/J, PL/J, and (SJL/J x PL/J)F1 mice. Influence of I-A haplotype on encephalitogenic epitope of myelin basic protein

Guinea pig basic protein (GPBP)-immune lymph node cells (LNC) from SJL, PL, and SJL x PL (F1) mice proliferated to whole GPBP and GPBP fragments 1-37, 43-88, and 89-169. All three strains of mice developed experimental allergic encephalomyelitis (EAE) by active immunization with whole GPBP or by passive transfer of LNC cultured with whole GPBP. SJL (H-2s) and PL (H-2u) mice developed EAE by active immunization with fragments 89-169 or 1-37, respectively, or by passive transfer of LNC cultured with the same Ag. F1 mice developed EAE by active immunization only with fragment 1-37 or by passive transfer of LNC cultured with either of the above fragments. Removal of macrophages (MO) from immune-F1 LNC resulted in the loss of a proliferative response and the ability to transfer EAE. Reconstitution of MO-depleted immune F1 T cells with either F1-, SJL-, or PL-MO restored the proliferative responses to whole GPBP and the three fragments. Cultures of immune F1 T cells reconstituted with any of the three MO populations and incubated with whole GPBP passively transferred EAE into naive F1 mice. Immune F1 T cells cultured with F1 MO in the presence of either fragment 1-37 or 89-169 transferred EAE. F1 T cells cultured with SJL MO were able to transfer EAE only if the Ag was fragment 89-169, whereas F1 T cells cultured with PL MO were able to transfer disease only if incubated in the presence of fragment 1-37. F1 mice are passively susceptible to EAE induced by adoptive transfer of cells reactive to either the N-terminal or C-terminal fragment and that the encephalitogenic determinant of GPBP is related to the genome of MO present in vitro.     

Adoptive transfer of experimental allergic encephalomyelitis in mice with the aid of pertussigen from Bordetella pertussis

Adoptive transfer of experimental allergic encephalomyelitis (EAE) in (SJL X BALB/c)F1 mice was accomplished by an iv injection of 2.4 to 4.7 X 10(7) lymph node cells (LNC) from mice immunized with mouse spinal cord emulsified in complete Freund's adjuvant when both donors and recipients had been treated iv with 400 ng of pertussigen at the time of immunization for the donors and on transfer of cells for the recipients. Pertussigen was essential in both donors and recipients for development of frank EAE. Signs of EAE in recipients were delayed, appearing 21-23 days after cell transfer; the maximum response at about Day 27 is considerably delayed in comparison with other reported studies on passive transfer of EAE. Histologically, recipient mice with paralysis due to EAE had typical perivascular infiltrates of mononuclear cells in the brain and spinal cord. The mechanisms by which pertussigen promotes the development of EAE after adoptive transfer of sensitized LNC are uncertain.     

Autoimmune effector cells. III. Role of adjuvant and accessory cells in the in vitro induction of autoimmune encephalomyelitis

The present investigation shows that autoreactive effector cells that transfer experimental allergic encephalomyelitis (EAE) can be activated from spleens and lymph nodes of Lewis rats given a single injection of 25 micrograms myelin basic protein (BP) in incomplete Freund's adjuvant (IFA), despite the fact that the cell donors do not develop EAE. Rather, these donor rats are unresponsive to EAE when given an encephalitogenic emulsion of BP in complete Freund's adjuvant (CFA). Lymphoid cells from rats given a single injection of BP-IFA were almost as effective as cells from BP-CFA-treated rats with respect to transferring EAE after in vitro activation with BP or concanavalin A (Con A). Irrespective of whether donors received BP in IFA or CFA, BP-cultured spleen and lymph node cells (SpC and LNC, respectively) transferred EAE, whereas Con A-cultured SpC but not LNC exhibited effector cell activity. Con A-cultured LNC were able to transfer EAE if the cultures were reconstituted with irradiated adherent phagocytic cells (which could be obtained from normal Lewis rat spleens) or with conditioned medium from these adherent SpC. These findings indicate that accessory cells are required for in vitro induction of this T cell-mediated autoimmune response.     

Regulation of experimental allergic encephalomyelitis. II. Appearance of suppressor cells during the remission phase of the disease

Lewis rats are susceptible to experimental autoimmune encephalomyelitis (EAE). Most rats recover from paralysis and are subsequently resistant to the disease. In an adoptive transfer system, we found that lymph node cells (LNC) from rats that had recovered from EAE protect syngeneic recipients from the disease when the latter are challenged with encephalitogenic myelin basic protein and adjuvant after receiving donor cells. Suppression is antigen-specific and requires viable LNC. In contrast to the suppressor cells we previously studied in tolerized rats, which were nonadherent T lymphocytes, the suppressor cells found in rats that have recovered from EAE adhere to glass wool. However, they are not retained on Sephadex G-10 columns to which macrophages adhere. Suppressor activity is enriched in the nylon wool-adherent LNC population (which consists of approximately 80% Ig+ cells). Our findings suggest that activation of adherent suppressor cells may be implicated in recovery from EAE. These may be adherent T cells, or B cells that produce anti-BP blocking antibodies.     

Immunosuppression of experimental allergic encephalomyelitis. III. In vitro evidence for induction of suppressor T lymphocytes in draining lymph node cells of animals immunized with myelin basic protein complexed to lipopolysaccharides

We recently demonstrated that Lewis rats immunized with bacterial lipopolysaccharides (LPS) precomplexed to guinea pig myelin basic protein (BP) in complete Freund's adjuvant were less effective in inducing experimental allergic encephalomyelitis (EAE) than BP-immunized controls. When tested in vitro both lymph node cells (LNC) and spleen cells (SpC) of animals immunized with BP-LPS were less effective in proliferative responses to various mitogens, which included phytohemagglutinin, concanavalin A, purified protein derivative of tuberculin, LPS, and BP. Of importance immunization of rats with BP complexed to LPS results in the generation of cells in lymph nodes of these animals that suppress the mitogenic response of BP-immunized LNC and also SpC in mixed lymphocyte cultures. The suppressive effect of these cells in mixed lymphocyte culture reaction was found specifically in response to BP and to a lesser extent to LPS in LNC. SpC of BP-LPS immunized animals did not suppress the proliferative response to SpC of BP-immunized animals. Treatment of these LNC with antithymocyte serum and complement abolished this suppressive effect of LNC, suggesting that the immunoregulatory cells in LNC of BP-LPS immunized animals are suppressor T lymphocytes. The parallel between the in vitro induction of suppressor T lymphocytes in the draining LNC and the function of LPS in the development of EAE in Lewis rats suggests a possible immunologic significance of the effect.     

Encephalitogenic and proliferative responses of Lewis rat lymphocytes distinguished by position 75- and 80-substituted peptides of myelin basic protein

The encephalitogenic and proliferative responses of Lewis rat lymphocytes were defined by use of synthetic peptide GP68-84, representing the 68-84 sequence of guinea pig myelin basic protein (GPMBP), and otherwise identical peptides containing substitutions of either A75 or P80 residues. The comparative activities of these peptides were tested in the following bioassays: 1) active induction of experimental autoimmune encephalomyelitis (EAE), 2) potentiation of EAE transfer activity by MBP- or peptide-sensitized lymph node cells (LNC), 3) in vitro proliferation of MBP- or peptide-sensitized LNC, and 4) in vitro proliferation of an encephalitogenic T cell line. The GP68-84 peptide exhibited potent activity in all four bioassays. In contrast, [A75]GP68-84 and [P80]GP68-84 exhibited a selective loss of certain activities while retaining activity in other bioassays. For example, LNC were activated by culture with [A75]GP68-84 to express potentiated EAE transfer activity. Furthermore, [A75]GP68-84 and GP68-84 were equipotent in stimulating the proliferation of the encephalitogenic T cell line. However, [A75]GP68-84 was virtually inactive in assays measuring the induction of EAE or the proliferation of either GPMBP- or [A75]GP68-84-sensitized LNC. Conversely, the [P80]GP68-84 peptide actively induced EAE and potentiated EAE cellular transfer activity but was incapable of stimulating proliferation of either GPMBP-sensitized LNC or an encephalitogenic T cell line. When [P80]GP68-84 was used for sensitization, in vitro proliferation of LNC was stimulated, but only by MBP sequences containing a P80 substitution. Overall, these results indicate that at least two structurally distinct T cell determinants of GP68-84 regulate functionally diverse encephalitogenic and proliferative activities of EAE-associated T cells.     

Enhanced transfer of experimental allergic encephalomyelitis with strain 13 guinea pig lymph node cells: requirement for culture with specific antigen and allogeneic peritoneal exudate cells

Previously, we reported that transfer of experimental allergic encephalomyelitis (EAE) with sensitized peritoneal exudate cells (PEC) in strain 13 guinea pigs is markedly enhanced if the cells are first cultured with specific antigen, myelin basic protein (BP). These cells also undergo considerable antigen-specific proliferation. In contrast, the data reported here show that lymph node cells (LNC) from sensitized animals display neither enhanced transfer nor antigen-specific proliferation after culture with BP. Enhanced transfer is obtained, however, if a second nonspecific signal is available. This second signal is provided by the presence of normal allogeneic strain 2 PEC in culture. After culture with BP and strain 2 PEC, 2.5 to 5 x 10(7) strain 13 LNC transfer disease reproducibly, in contrast with approximately 1 x 10(9) previously required for successful transfer. Addition of allogeneic or syngeneic PEC without antigen does not lead to enhanced transfer by LNC. Culture with normal syngeneic PEC plus BP oly infrequently enhances transfer by LNC. The intense mixed lymphocyte reaction (MLR) induced by addition of strain 2 PEC to strain 13 LNC precludes the use of 3H-TdR incorporation for detection of proliferation by EAE effector cells. However, inhibition of transfer with low doses of mitomycin C (2 to 5 micrograms/ml) pluse the fact that EAE effector cells are found almost exclusively in the light fraction of BSA gradients after (but not before) culture suggests that the latter are induced to proliferate in culture.     

Interleukin 1 and myelin basic protein synergistically augment adoptive transfer activity of lymphocytes mediating experimental autoimmune encephalomyelitis in Lewis rats

This investigation focused on the role of adherent accessory cells and their cellular product, interleukin 1 (IL 1), in cellular immune responses associated with experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Guinea pig myelin basic protein (GPMBP)-sensitized lymph node cells (LNC) responded in culture with GPMBP by undergoing activation as measured by augmented transfer of EAE to syngeneic recipients, and proliferation as measured by [3H]thymidine incorporation. GPMBP-sensitized LNC, after depletion of adherent accessory cells, no longer responded to GPMBP in the EAE transfer activation assay. In contrast, aliquots of the same LNC preparation exhibited proliferative responses to GPMBP that were only partially reduced. Addition of irradiated thymocytes to adherent cell-depleted cultures fully reconstituted responsiveness to GPMBP in the activation assay and restored full reactivity to GPMBP in the proliferation assay. Furthermore, addition of either purified human IL 1 or recombinant human IL 1 to adherent cell-depleted cultures reconstituted reactivity to GPMBP in the EAE transfer activation assay and augmented GPMBP-specific proliferative responses. Anti-Ia monoclonal antibodies blocked GPMBP + IL 1-induced cellular activation of nonadherent LNC. These results demonstrate that both IL 1 and Ia molecules are important in the pathway leading to GPMBP-induced activation of EAE-inducing T lymphocytes. Furthermore, these results suggest that different accessory signals may be required for optimal induction of GPMBP-induced lymphocyte activation vs GPMBP-specific proliferative responses.     

Lymphocytes from SJL/J mice immunized with spinal cord respond selectively to a peptide of proteolipid protein and transfer relapsing demyelinating experimental autoimmune encephalomyelitis.

Relapsing experimental autoimmune encephalomyelitis (R-EAE) can be induced in SJL/J mice by immunization with spinal cord homogenate and adjuvant. The specific Ag(s) responsible for acute disease and subsequent relapses in this model is unknown. Myelin basic protein (BP), an encephalitogenic peptide of BP (BP 87-99), and proteolipid protein (PLP) can each induce R-EAE in SJL/J mice, and a peptide of PLP (PLP 139-151) has been reported to induce acute EAE. To determine the encephalitogens in cord-immunized mice with R-EAE, the in vitro proliferative responses of lymph node cells (LNC) and central nervous system mononuclear cells to BP, BP peptides, and PLP peptides were examined during acute EAE and during relapses. LNC responded only to PLP peptides 139-151 and 141-151 and did not respond to BP or its peptides during acute or chronic disease. Central nervous system mononuclear cells also preferentially responded to PLP 139-151 and 141-151 during acute and relapsing disease. A PLP 139-151 peptide-specific Th cell line was selected from LNC of cord-immunized donors. Five million peptide-specific line cells transferred severe relapsing demyelinating EAE to naive recipients. We conclude that PLP peptide 139-151 is the major encephalitogen for R-EAE in cord-immunized SJL/J mice. We demonstrate for the first time that Th cells specific for this peptide are sufficient to transfer relapsing demyelinating EAE. The predominance of a PLP immune response rather than a BP response in SJL/J mice suggests that genetic background may determine the predominant myelin Ag response in human demyelinating diseases such as multiple sclerosis.     

Cellular transfer of experimental allergic encephalomyelitis: Altered disease pattern in irradiated recipient Lewis rats

Irradiation of recipient Lewis rats 6–24 hr prior to injection of sensitized lymph node cells (LNC) altered the pattern of transferred experimental allergic encephalomyelitis (EAE). Recipients subjected to total body irradiation in doses ranging from 500 to 1000 rads developed paralysis; nonirradiated control recipients did not do so. Histopathologic changes of EAE, in terms of number of descrete cellular infiltrates, were potentiated in the total body irradiated recipients. Among LNC recipients subjected to regional irradiation (850 rads) of the head or lower spinal column, paralysis was observed only in those animals where the irradiation impinged upon the spinal cord. Cellular infiltrates of EAE were numerically more common in the irradiated region of the neuraxis. The findings are discussed in terms of irradiation rendering the central nervous system of animals and man more vulnerable to autoimmune injury.     

Role of macrophage-myelin basic protein interaction in the induction of experimental allergic encephalomyelitis in Lewis rats

Stimulated peritoneal exudate cells (PEC) containing activated macrophages (M phi) of Lewis (Le) rats were exposed for 1 hr in vivo or in vitro to radioiodinated soluble myelin basic protein (MBP) or MBP incorporated into magnetic microspheres (MBP-microspheres). The uptake by M phi of the dose of microsphere-bound MBP averaged 6.2%, whereas the average uptake of soluble MBP was 0.17%. Naive rats were sensitized with M phi-associated MBP or M phi-associated MBP-microspheres via the hind footpads without the aid of conventional immunologic adjuvants. Draining lymph node cells (LNC) or spleen cells from sensitized rats were cultured for 3 days with guinea pig MBP (GPMBP) alone or in combination with concanavalin A (Con A), then injected i.v. into naive recipients. Clinical signs of experimental allergic encephalomyelitis (EAE) appeared 6 days after transfer of LNC or spleen cells, and typical CNS lesions were seen in recipients sacrificed 10 to 14 days after transfer. The challenge of MO-MBP-sensitized rats with MBP-CFA resulted in severe clinical signs of EAE marked by an accelerated onset of neurologic symptoms.     

Adoptive transfer of experimental allergic encephalomyelitis in SJL/J mice after in vitro activation of lymph node cells by myelin basic protein: requirement for Lyt 1+ 2- T lymphocytes

Optimal conditions were established for the adoptive transfer of experimental allergic encephalomyelitis (EAE) in SJL/J mice. Lymph node cells from SJL/J mice primed in vivo with myelin basic protein (BP) were incubated in vitro with BP. These cells proliferated specifically to BP and when transferred at the optimal conditions into syngeneic mice induced EAE in 100% of the recipients. The in vitro proliferative response to BP was dependent on the presence of Lyt 1+ 2- T lymphocytes. Furthermore, when the activated LNC were treated before transfer with anti-Thy 1 or anti-Lyt 1 antibody and C, neither clinical nor histologic signs were observed in the recipients, whereas treatment with anti-Lyt 2 antibody and C had no effect. These results indicated that Lyt 1+ 2- T cells are responsible for the transfer of EAE.     

Enhanced proliferation,survival, and yield of cultured myelin basic protein (MBP)-reactive Lewis rat lymph node cells following dual activation with concanavalin A and MBP

Lymph node cells (LNC) from Lewis rats sensitized 7 days previously to myelin basic protein (MBP) exhibit greatly increased survival and undergo much greater proliferation when cultured in the presence of both concanavalin A (Con A) and MBP. Dual activation, in contrast to culture with either the mitogen or the neural antigen, results in a substantially greater yield of lymphoblasts with the capacity to transfer experimental allergic encephalomyelitis (EAE) to normal syngeneic recipients. Dual activation provides a practical advantage over other methods reported to date for securing larger numbers of functional MBP-reactive lymphoid cells for immunologic studies.     

Soluble factors in tolerance and contact sensitivity to DNFB in mice. VII. Characterization of a monoclonal, efferent-acting suppressor factor with specificity for DNP/H-2Kd

To study further soluble factors which regulate contact sensitivity (CS) to 2,4-dinitrofluorobenzene (DNFB), hapten-primed spleen cells from BALB/c mice were used to make T-cell hybridomas. A hybrid constitutively producing a suppressor factor was identified and cloned (clone 3-10). Incubation of BALB/c DNFB immune lymph node cells (LNC) in the 3-10 supernatant suppressed the ability of the immune cells to transfer CS to DNFB. The passive transfer of CS to oxazalone or to 2,4,6-trinitrochlorobenzene (TNCB) was not suppressed by the 3-10 factor. The hapten specificity of the 3-10 factor further was demonstrated by the ability of DNFB immune LNC but not LNC from unsensitized or from TNCB-sensitized mice to absorb the factor. The 3-10 factor also was adsorbed by DNFB-immune LNC from mice that were syngeneic with BALB/c mice at the K locus of the MHC (e.g., B10.D2 and D2.GD). Pretreatment of DNFB-immune LNC with monoclonal anti-Kd antibody or with anti-DNP antibodies blocked the ability to adsorb the factor. These results indicated that the 3-10 suppressor factor binds to DNP/H-2Kd complexes on immune LNC. Nylon wool-purified T cells (83% Thy-1.2+) from DNFB-immune LNC were able to adsorb the factor as well as unseparated immune LNC. Furthermore, treatment of immune LNC with anti-Thy-1.2 plus C' abrogated the ability of the cells to adsorb the factor, indicating that the cellular target of the 3-10 factor is a T cell. In addition, treatment of the immune LNC with an autoantiidiotypic antiserum (CS 231) plus C', which depletes DNP-specific delayed-type hypersensitivity effector T (TDH) cells, also abrogated the ability of the cells to adsorb the factor. Finally, the suppressor factor was adsorbed and eluted from DNP affinity columns but was not adsorbed by TNP affinity columns. Collectively, these results indicate that although the monoclonal 3-10 suppressor factor has affinity for DNP, focusing of the factor on the TDH cells requires recognition of DNP in the context of the appropriate MHC determinant, Kd.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. III. Cholera enterotoxin induces lymph node cells to release soluble factor(s) which enhance(s) antibody synthesis by antigen-treated lymph node cells.

Unprimed or KLH-primed rabbit lymph node cells were pulsed with cholera enterotoxin or KLH for 2 hr and washed. KLH-treated LNC were mixed with equal numbers of CT-treated LNC or boiled CT-treated LNC. Cocultivation of CT-treated LNC with KLH-treated cells resulted in at least a 100% increase in antibody synthesis compared to control cultures. Delaying cocultivation for 24 hr reduced enhancement to 25%. Thus it appears that an early event—before 24 hr—is involved in CT enhancement. Using ¹²⁵I-CT, it was shown that these effects were not due to CT carry-over. When KLH- and CT-pulsed LNC were cultured in chambers separated by polycarbonate membranes (0.2- to 0.4-μm pore size) antibody production was enhanced 50–80%. Supernates of CT-treated LNC also enhanced antibody production by KLH-treated LNC. These results suggest that CT triggers the release of soluble factor(s) which enhance(s) antibody synthesis by antigen-primed and antigen-challenged LNC.     

Murine delayed hypersensitivity (DHS): modulation of in vitro antigen-induced responsiveness of lymph node cells from mice expressing DHS to human gamma-globulin by lymphocyte populations from normal syngeneic animals

Using a chemically defined, protein-free medium, the modulatory effect of normal (N) lymphocytes on in vitro antigen-induced proliferation by lymph node cells (LNC) from mice immunized to express delayed hypersensitivity (DHS) to human γ-glogulin (HGG) was quantitated in coculture. LNC from normal syngeneic animals exerted little if any effect on immune-LNC proliferation. Compared with immune-LNC plus N-LNC coculture response. N thymus cells (TC) were consistently suppressive while N spleen cells (SC) varied in their effect from a marginal to a marked potentiation of radiolabeled thymidine incorporation. Inactivation of N-SC suspensions by X irradiation prior to coculture with immune LNC abrogated the increased responsiveness. It therefore appeared that interaction of immune LNC and antigen resulted in recruitment of N-SC to proliferate. Separation of N-SC suspensions to provide enriched populations of thymic-independent (B) and thymic-dependent (T) lymphocytes showed that B cells augmented and T cells suppressed HGG-induced incorporation of [3H] thymidine when cocultured with immune LNC.     

Spontaneous cell-mediated cytotoxicity (SCMC) and antibody-dependent cellular cytotoxicity (ADCC) in peripheral blood and draining lymph nodes of patients with mammary carcinoma

Summary In 14 patients with primary invasive mammary carcinoma (T_1–3N_0–1M₀) lymphocyte preparations obtained from peripheral blood (PBL) and tumor-free or metastatic lymph nodes (LNC) were examined for spontaneous (SCMC) and antibody-dependent cellular cytotoxicity (ADCC) against the allogeneic melanoma cell line IGR3 and a thymoma cell line THY. The cytotoxic activities were compared with those of PBL from healthy women and of LNC from normal mesenteric lymph nodes. In addition, the percentages of E-, EA-, EAC-rosette-forming cells and of surface Ig (SIg)-positive cells were determined for all PBL and LNC suspensions tested.As a rule, LNC exhibited significantly lower SCMC and ADCC than the corresponding PBL preparations. The difference was particularly pronounced in ADCC assays, due to a strikingly low K-cell activity of LNC cells. Consonant with this observation was a reduced percentage of Fc-receptor-bearing cells in LNC suspensions. In SCMC assays the IRG3 targets used in three tests appeared to be less susceptible to LNC effectors than THY targets. No difference in cytotoxicity was noted between PBL from breast cancer patients and from normal women; nor did LNC from tumor-free or metastatic axillary nodes and normal LNC from mesenteric nodes show a significantly different degree of SCMC and ADCC. Abbreviations used in this paper: ADCC, antibody dependent cellular cytotoxicity; E, neuraminidase treated sheep red blood cells; EAox, ox erythrocytes sensitized with rabbit-anti-ox IgG; EAh, AB erythrocytes sensitized with anti M and N antiserum; EAC, ox erythrocytes sensitized with rabbit-anti-ox IgM and sublytic human complement; LNC, lymph node cells; MEM-FCS, minimal essential medium supplemented with heated fetal calf serum, antibiotics, etc.; NK, de]natural killing; PBL, peripheral blood lymphocytes; PBS, phosphate-buffered saline; SIg, surface immunoglobulin; SCMC, spontaneous cell-mediated cytotoxicity     

The intracellular oxidation of 2',7'-dichlorofluorescin in murine T lymphocytes

Intracellular reactive oxygen species (ROS) production by activated murine T lymphocytes was investigated by analyzing intracellular dichlorofluorescin (DCFH(2)) oxidation in lymph node cells (LNC). An increase in DCFH(2) oxidation in LNC induced by phorbol myristate acetate (PMA) was detected by flow cytometry. It was confirmed that this increase was present in Thy1(+) LNC. We examined the contribution to intracellular DCFH(2) oxidation of ROS released by leukocytes other than T cells present in the LNC suspension. Superoxide dismutase, catalase, and glutathione/glutathione peroxidase inhibited the PMA-induced increase in intracellular DCFH(2) oxidation. Furthermore, PMA failed to elicit DCFH(2) oxidation in LNC isolated from mice lacking a functional NADPH oxidase (gp91(phox) gene knockout mice), but this response could be restored in these cells by the addition of T cell-depleted LNC from wild-type litter mates. This study highlights the necessity for caution in using the DCFH(2) assay to demonstrate specific intracellular ROS production in heterogeneous cell populations. It also suggests that cells other than T cells in lymph node populations may, through production of ROS, influence the intracellular redox state of T lymphocytes.