Interleukin 10 and interferon gamma regulation of experimental Trypanosoma cruzi infection.

Studies were undertaken to determine whether interleukin 10, (IL-10) a cytokine shown to inhibit interferon gamma (IFN-gamma) production, was involved in Trypanosoma cruzi infections in mice. Exogenous IFN-gamma protects mice from fatal infection with T. cruzi. Furthermore, resistant B6D2 mice developed fatal T. cruzi infections when treated with neutralizing anti-IFN-gamma monoclonal antibody (mAb). Thus, endogenous as well as exogenous IFN-gamma is important in mediating resistance to this parasite. Because both T. cruzi-susceptible (B6) and -resistant (B6D2) mouse strains produced IFN-gamma during acute infection, we looked for the concomitant production of mediators that could interfere with IFN-gamma-mediated resistance to T. cruzi. We found that IL-10-specific mRNA was produced in the spleens of mice with acute T. cruzi infections. In addition, spleen cell culture supernatants from infected B6 mice, and to a lesser extent B6D2 mice, elaborated an inhibitor(s) of IFN-gamma production. This inhibitor(s) was neutralized by anti-IL-10 mAb. These experiments demonstrated the production of biologically active IL-10 during T. cruzi infection. In further studies in vitro, it was shown that IL-10 blocked the ability of IFN-gamma to inhibit the intracellular replication of T. cruzi in mouse peritoneal macrophages. Thus, in addition to its known ability to inhibit the production of IFN-gamma, IL-10 (cytokine synthesis inhibitory factor), may also inhibit the effects of IFN-gamma. These experiments demonstrate that IL-10 is produced during infection with a protozoan parasite and suggest a regulatory role for this cytokine in the mediation of susceptibility to acute disease.

A metalloprotease inhibitor blocks shedding of the 80-kD TNF receptor and TNF processing in T lymphocytes.

TNF is synthesized as a 26-kD membrane-anchored precursor and is proteolytically processed at the cell surface to yield the mature secreted 17-kD polypeptide. The 80-kD tumor necrosis factor (TNF) receptor (TNFR80) is also proteolytically cleaved at the cell surface (shed), releasing a soluble ligand-binding receptor fragment. Since processing of TNF and TNFR80 occurs concurrently in activated T cells, we asked whether a common protease may be involved. Here, we present evidence that a recently described inhibitor of TNF processing N-(D,L-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl)L- 3-(2'naphthyl)- alanyl-L-alanine, 2-aminoethyl amide (TAPI) also blocks shedding of TNFR80, suggesting that these processes may be coordinately regulated during T cell activation. In addition, studies of murine fibroblasts transfected with human TNFR80, or a cytoplasmic deletion form of TNFR80, reveal that inhibition of TNFR80 shedding by TAPI is independent of receptor phosphorylation and does not require the receptor cytoplasmic domain.

Ultrafast electron diffraction from nanophotonic waveforms via dynamical Aharonov-Bohm phases.

Electron interferometry via phase-contrast microscopy, holography, or picodiffraction can provide a direct visualization of the static electric and magnetic fields inside or around a material at subatomic precision, but understanding the electromagnetic origin of light-matter interaction requires time resolution as well. Here, we demonstrate that pump-probe electron diffraction with all-optically compressed electron pulses can capture dynamic electromagnetic potentials in a nanophotonic material with sub-light-cycle time resolution via centrosymmetry-violating Bragg spot dynamics. The origin of this effect is a sizable quantum mechanical phase shift that the electron de Broglie wave obtains from the oscillating electromagnetic potentials within less than 1 fs. Coherent electron imaging and scattering can therefore reveal the electromagnetic foundations of light-matter interaction on the level of the cycles of light.

Fully human CD19-specific chimeric antigen receptors for T-cell therapy.

Impressive results have been achieved by adoptively transferring T-cells expressing CD19-specific CARs with binding domains from murine mAbs to treat B-cell malignancies. T-cell mediated immune responses specific for peptides from the murine scFv antigen-binding domain of the CAR can develop in patients and result in premature elimination of CAR T-cells increasing the risk of tumor relapse. As fully human scFv might reduce immunogenicity, we generated CD19-specific human scFvs with similar binding characteristics as the murine FMC63-derived scFv using human Ab/DNA libraries. CARs were constructed in various formats from several scFvs and used to transduce primary human T-cells. The resulting CD19-CAR T-cells were specifically activated by CD19-positive tumor cell lines and primary chronic lymphocytic leukemia cells, and eliminated human lymphoma xenografts in immunodeficient mice. Certain fully human CAR constructs were superior to the FMC63-CAR, which is widely used in clinical trials. Imaging of cell surface distribution of the human CARs revealed no evidence of clustering without target cell engagement, and tonic signaling was not observed. To further reduce potential immunogenicity of the CARs, we also modified the fusion sites between different CAR components. The described fully human CARs for a validated clinical target may reduce immune rejection compared with murine-based CARs.

Quantitation of cytokine mRNA levels utilizing the reverse transcriptase-polymerase chain reaction following primary antigen-specific sensitization in vivo--I. Verification of linearity, reproducibility and specificity.

The role of cytokines in vivo has been difficult to assess. This difficulty is due, in part, to the limited number of producer cells and the strict regulation of cytokine production. In order to address this situation, we have developed assays which allow us to quantitate both protein production and steady state mRNA levels from specific in vivo sites. In this report, we present data utilizing these assays on cells obtained from draining LN following specific sensitization with antigen in vivo. In order to determine the relative quantities of cytokine mRNA, we modified the reverse transcriptase-polymerase chain reaction which had been previously described. The modified assay is (1) linear over a large concn range of input template (2) demonstrates a high degree of reproducibility (SE approximately 13%) and (3) is very sensitive. Utilizing this assay, we have measured a constitutive mRNA (DHFR), quantitated both the presence of lymphokine mRNA (IL-2) and the induction of cytokine mRNA (TNF alpha). In this report we have examined the kinetics of TNF alpha mRNA expression and have demonstrated that following epicutaneous sensitization with picryl chloride, there is rapid induction (within 24 hr) of TNF alpha mRNA in the draining LN and that the levels of mRNA remain detectable through d7. In addition, we determined the time course of production of TNF protein by the draining LN cells and found that it was similar to that of the mRNA levels. A potential pathologic role for immune response generated TNF alpha is also discussed. We believe these experiments demonstrate that cytokine production following antigen-specific sensitization in vivo can be analyzed at both the cellular and molecular level. The data suggests that this approach can be used to study cytokine regulation in vivo.

Further evidence for a common mechanism for shedding of cell surface proteins.

Pro-TNF alpha, Steel factor, type II IL-1R and IL-2R alpha were expressed in COS-7 cells and the generation of their soluble forms was examined. The release of all four proteins was strongly stimulated by the phorbol ester PMA and completely blocked by a hydroxamate-based inhibitor of metalloproteases. COS-7 cell membranes were found to cleave various synthetic pro-TNF alpha peptides with the same specificity as a partially purified TNF alpha converting enzyme purified from human monocytic cells, suggesting that the same enzyme may be responsible for at least some of the COS-7 cell shedding activity.

In vitro detection of lymphokines produced by in vivo activated lymphocytes.

The purpose of these experiments was to develop a method to measure the production of lymphokines by cells which were activated by antigen in vivo. Previous protocols have been relatively unsuccessful since small, if any, amounts of lymphokines were available for measurement when in vivo antigen activated lymphocytes (AAL) were examined. These unsuccessful experiments usually employed supernatants derived from in vivo AAL which had been cultured in vitro for 4-24 h. As an alternative to assaying supernatants for the presence/absence of lymphokines, we have developed a co-culture system in which the indicator cells are directly added to the wells containing the in vivo AAL. Utilizing this system in conjunction with appropriate neutralizing monoclonal antibodies, we have demonstrated that interleukin-2, interleukin-3/colony stimulating factor and tumor necrosis factor-alpha can be readily detected from in vivo AAL.

Lymphokine production by MLR-reactive reaction lymphocytes obtained from normal mice and mice rendered tolerant of class II MHC antigens.

A large proportion of mice rendered neonatally tolerant of class II MHC antigens respond to the tolerogen in vitro in an MLR, while simultaneously maintaining tolerance in vivo as evidenced by acceptance of a skin graft bearing the tolerated antigens. To determine whether this discrepancy between in vivo and in vitro tolerance is reflective of differences in the amount and/or type of lymphokines produced by tolerant lymphocytes, we have examined the ability of tolerogen-reactive lymphocytes to produce IL-2, IL-4/5, and IFN in vitro in an MLR. Our results demonstrate that when stimulated with the tolerogen, lymphocytes from both normal and tolerant responders produce IL-2 and interferon. However, in comparison to normal cells, 2 alterations in the tolerogen-specific responses of lymphocytes obtained from tolerant mice were identified. (1) The amount of IL-2 in the supernatants derived from tolerant cultures declines prematurely compared to normal cultures. This premature decline in IL-2 production was due neither to a lower frequency of Th cells as judged by limit dilution analysis nor to an increase in IL-2R expression on tolerant lymphocytes as measured by FACS analysis. (2) IL-4 and presumably IL-5 can be demonstrated in supernatants of tolerant, but not normal, lymphocytes stimulated by the tolerogen. Thus although lymphocytes from MLR-positive tolerant mice generate substantial quantities of lymphokines in response to the tolerogen, the pattern of lymphokine production is unusual when compared to that of normal lymphocytes. These results are inconsistent with the notion that a global lack of helper activity, per se, is responsible for the maintenance of tolerance in these mice and furthermore suggest that tolerance could be the result of "inappropriate" lymphokine production.

Differential expression of helper versus effector activity in mice rendered neonatally tolerant of class II MHC antigens.

Since tolerogen-specific helper activity is present in MLR-positive class II MHC tolerant mice, a loss of helper activity is unlikely to be responsible for the maintenance of tolerance in these mice. An alternative hypothesis, that effector cell function is selectively down-regulated, has been examined with lymphocytes from MLR-positive class-II MHC tolerant mice on both the A strain and the B10 background. The results demonstrate that lymphocytes from A-strain-tolerant mice were unable to generate tolerogen-specific effector cells in any of the assays tested (CML with or without exogenous growth factor and DTH following in vivo priming or local adoptive transfer), even though these mice possess tolerogen-responsive T helper cells. In contrast, a majority of MLR-positive tolerant mice on the B10 background generated measurable tolerogen-specific cytotoxic activity in the absence of exogenous growth factor, and all the mice examined generated substantial cytotoxic activity in the presence of exogenous growth factor. However, in a local adoptive transfer reaction, lymphocytes from these mice failed to display DTH. It is concluded that tolerance is maintained by selective impairment of class II specific effector functions and that regulation of DTH rather than CTL activity may be central to maintenance of in vivo tolerance to class II MHC antigens.

Biological effects and fate of a soluble, dimeric, 80-kDa tumor necrosis factor receptor in renal transplant recipients who receive OKT3 therapy.

A preliminary clinical study of renal allograft recipients revealed that a dimeric form of the human 80 kDa soluble receptor (sTNFR:Fc) for tumor necrosis factor (TNF) is well tolerated and attenuates the OKT3-induced acute clinical syndrome. The current study determined the in vivo biological effects and fate of sTNFR:Fc in these patients. Serial assessment of both antigenic and biological activities of circulating TNF and sTNFR:Fc have led to the following observations. (1) Although control patients typically responded to the first OKT3 injection with a rapid increase of biologically active TNFalpha, patients on sTNFR:Fc therapy had markedly higher serum TNFalpha antigenic levels, but no detectable bioactivity. Thus, sTNFR:Fc functioned as a potent antagonist, despite its cytokine-carrier effect. (2) Peak sTNFR:Fc levels averaging 800 and 2500 ng/ml were routinely achieved in vivo, using the low-dose (0.05 mg/kg) and high-dose (0.15 mg/kg) protocols. (3) The half-life of circulating sTNFR:Fc was estimated to be approximately 4.4 days, and levels of p80 receptors in treated patients remained significantly above those in control patients for at least 20 days. (4) In vitro blocking studies demonstrated that circulating sTNFR:Fc remained biologically active for 2 weeks. These results demonstrate that under current protocols, significant serum levels of sTNFR:Fc, capable of effectively neutralizing TNF activity over prolonged periods, can be achieved. The persistent OKT3 side effects observed, despite sTNFR:Fc therapy, are therefore likely to be caused by factors other than TNF.

Evaluation of recombinant human soluble dimeric tumor necrosis factor receptor for prevention of OKT3-associated acute clinical syndrome.

Tumor necrosis factor alpha (TNFa) has been shown to be the primary cytokine responsible for the OKT3-induced acute clinical syndrome (OKT3-ACS). Recombinant human soluble tumor necrosis factor receptor (TNFR:Fc) is a dimer of the p80 TNF receptor, which binds both TNFa and lymphotoxin (LT). Renal allograft recipients undergoing OKT3 therapy for steroid-resistant rejection were randomized to receive OKT3 alone or in combination with TNFR:Fc to determine its safety and efficacy in decreasing the severity of OKT3-ACS and in restoring renal function. Six of 12 patients were given TNFR:Fc prior to each of the first two injections of OKT3. All patients were monitored for manifestations of OKT3-ACS and changes in renal function. In addition, serial serum samples were assayed for TNFa and TNFR:Fc levels (ELISA) and TNFa bioactivity (L929). No adverse side effects were identified in patients receiving TNFR:Fc. Patients treated with TNFR:Fc had significantly fewer symptoms by day 2 of OKT3, and had a lower overall incidence of chills and arthralgias. Renal dysfunction reversed within 24 hr in the TNFR:Fc-treated group in contrast to the 48-72-hr delay in the control group. Antigenic TNFa levels increased in the control group from < 10 pg/ml pre OKT3 to a mean peak level of 30 +/- 13 pg/ml on day 1 and decreased to pretreatment levels by day 2. TNFR:Fc-treated patients had a mean peak TNFa level of 235 +/- 135 pg/ml, suggesting a carrier effect of TNFR:Fc. In contrast, bioactivity was barely detectable (mean 20 +/- 14 pg/ml) in the day 1 samples from TNFR:Fc-treated patients, whereas significant bioactivity (peak mean 60 +/- 35 pg/ml) was detected in sera from control patients. TNF receptor levels reached 600 ng/ml in treated patients and remained elevated for up to 18 days confirming the long half-life of TNFR:Fc. This phase 1 trial demonstrates that TNFR:Fc is well tolerated and may limit the severity of OKT3-ACS. The most significant observation was a more rapid improvement in renal function in the TNFR:Fc-treated patients. The absence of TNFa bioactivity indicates that TNFR:Fc functions as a TNF antagonist. Further evaluation of higher doses of TNFR:Fc in OKT3-treated patients is currently in progress.

Mechanisms of neonatal transplantation tolerance.

Although our analysis is very incomplete, our data indicate that a complete explanation of tolerance induced neonatally to H-2 encoded alloantigens will include elements of clonal deletion and active suppression. Background genes not encoded in H-2 play an important role in determining the extent to which clonal reduction occurs. In mice of B10 background genotype, extensive elimination of clones capable of recognizing class I and class II antigens is accomplished. Tolerance to class I alloantigens alone seems to be maintained almost exclusively by this mechanism. In B10 background mice tolerant of class II antigens alone or in consort with class I alloantigens, peripherally maintained (post-thymic) active suppression exists, that is dependent upon donor I-J expression, and is superimposed on clonal reduction. As a consequence, the activities of class II alloantigen-specific cells appear to be reduced to background, a level insufficient to activate specific effector precursors, even in the presence of relevant alloantigens. Donor alloantigens expressed on chimeric cells that exist among central (bone marrow, thymus) and peripheral (spleen, lymph nodes) lymphoid cells are undoubtedly crucial both to the process of ongoing clonal elimination as well as of active suppression. The role of clonal reduction in the maintenance of class II alone tolerance in mice of A/J background is more obscure. If the class II alloreactive cells that provide helper/inducer activity are identical to the cells that provide effector function (cytotoxic T cells, TDTH cells), then clonal reduction seems not to occur. Instead, class II tolerogen reactive cells remain within the peripheral lymphoid tissues of the tolerant mice, but appear to be incapable of differentiating beyond the initial steps of antigen-driven activation. We know little about the process by which such cells could be placed under these differentiation strictures, nor why they are retained within the tolerant animal's lymphoid mass. However, the absence of detectable chimerism in alloclass II tolerant animals of A/J background may offer an important clue to solving this mystery.

[The problem of nitrate and nitrite in human food. II. The occurrence of nitrate, nitrite, and thiocyanate in human saliva (author's transl)]. / Das Nitrat-/Nitritproblem in der menschlichen Nahrung. II. Vorkommen von Nitrat, Nitrit and Thiocyanat im menschlichen Speichel.

When the alimentary nitrate intake is in the range of 50 mg NO3- the nitrate values in saliva of adults and children follow a Gaussian normal distribution. With high and very high nitrate intakes the individual fluctuations of nitrate in saliva are distributed randomly in a wide range with a factor of 1:4. The nitrite values in saliva fluctuate even more, so that a proper judgment should be made only in individual cases. The average content of thiocyanate in saliva of adults was found to be 134 ppm SCN-, the saliva of children contained 50 ppm SCN-. The estimated molecular ratio of thiocyanate to nitrite in saliva of adults and children may fluctuate between 8:1 and 1:1 depending on the nitrate content of food.

Differential production of IL-2 and IL-4 mRNA in vivo after primary sensitization.

The study of lymphokines has been almost entirely conducted by utilizing in vitro assay systems, long term cell lines, and clones. Thus, little information is available concerning the production of lymphokines/cytokines in vivo after specific antigenic stimulation. In order to address this limitation, we have modified the mRNA phenotyping system to allow for the quantitation of lymphokine mRNA after antigenic stimulation in vivo. We report here the production of both IL-2 and IL-4 mRNA in vivo after primary sensitization with picryl chloride. However, the time course of IL-2 and IL-4 mRNA production is discordant. The majority of IL-2 mRNA expression occurs from 1 to 3 days after antigenic exposure, whereas IL-4 mRNA expression occurs mainly from day 3 through day 5. Thus, the production in vivo of these two lymphokine mRNA after sensitization with picryl chloride appears to occur as a "cascade." These results 1) demonstrate that IL-4 mRNA is induced during a primary immune response in vivo and 2) raise the possibility that the generation of an immune response in vivo may involve a specific sequential production of certain lymphokines.

Tolerance to class II major histocompatibility complex molecules is maintained in the presence of endogenous, interleukin-2-producing, tolerogen-specific T lymphocytes.

We have examined the requirement for clonal reductions of tolerogen-reactive lymphocytes in mice of the A strain background rendered neonatally tolerant of class II major histocompatibility complex molecules. Tolerogen-specific mixed lymphocyte reactivity of lymphocytes obtained from 130 adult, class II tolerant mice, bearing a healthy skin allograft, was examined. Lymphocytes obtained from 86 mice responded to the tolerogen, in vitro, with a positive mixed lymphocyte response (MLR) indicating that a large proportion (75%) of adult class II tolerant mice on the A strain background are not clonally deleted for tolerogen-reactive lymphocytes. In addition, lymphocytes from 29 mice were MLR-negative to the tolerogen, and lymphocytes from 15 mice demonstrated such high amounts of proliferation to syngeneic stimulators that their specific response to the tolerogen could not be determined. In view of the discordance between the in vivo and in vitro expressions of tolerance in the MLR-positive mice, lymphocytes from these mice were compared with normal lymphocytes by several assays. 1) Tolerogen-specific proliferative responses obtained from both normal and tolerant lymphocytes could be inhibited by the addition of monoclonal antibodies specific for the relevant class II antigens; 2) quantitative differences in the ability of normal, as compared with tolerant cells, to respond to the tolerogen in the MLR were not apparent; 3) no evidence of qualitative differences in the cell-surface phenotype of the proliferating cell was observed, (i.e., the cells were Thy-1+, L3T4+, Lyt-2-); and 4) lymphocytes from both normal and MLR-positive tolerant mice produced substantial amounts of interleukin-2 in response to the tolerogen. Thus, clonal deletion of helper cells is not required for tolerance to class II major histocompatibility complex antigens and we propose that tolerance may be maintained by either 1) in vivo suppression of the tolerogen-specific helper cells or 2) selective deletion or suppression of class II specific effector cells.

Allo-I-J determinants participate in maintenance of neonatal H-2 tolerance.

To evaluate the role of IJ antigens in maintenance of the tolerant state in adult H-2 tolerant mice, we have attempted to abolish tolerance by injecting monoclonal antibodies (mab) specific for host, donor, or third party IJ antigens into adult H-2 tolerant mice. Abolition of tolerance was evidenced by the rejection of fresh test skin grafts bearing the tolerated antigens. Whole H-2 tolerant mice treated with anti-IJ mab specific for donor (allo) IJ antigens rejected their test skin grafts, indicating that tolerance had been abolished. When two other types of tolerant mice were tested, we found that mice tolerant of class II antigens alone, but not mice tolerant of an IJ thru D disparity, were susceptible to the anti-donor IJ mab treatment. In addition, adult tolerant mice were unaffected by treatment with either anti-host or anti-third party IJ mab. When tested in vitro, lymphoid cells from tolerant mice, the tolerance of which was abolished by anti-IJ mab, remained unresponsive to the tolerogen, just as untreated (control) tolerant mice, in several in vitro assays (e.g., mixed lymphocyte reaction, cytotoxic T cell precursor frequency and bulk cell-mediated lysis without growth factor). Mice treated with antidonor IJ mab, however, unlike mice treated with anti-host or third party IJ mab, were capable of generating tolerogen-specific T cells in the absence of exogenous growth factor. Thus in the strain combinations we used, adult mice tolerant of either the entire H-2 region or of the class II major histocompatibility complex region alone are susceptible to abolition of the tolerant state by treatment with anti-donor IJ mab. Coincidentally, lymphoid cells from these mice generate sufficient endogenous T helper activity to activate the tolerogen-specific cytotoxic T cells. We suspect that these latter cells may be responsible for rejection of grafts bearing the tolerated antigens.

Analysis of cytokine mRNA expression in the central nervous system of mice with experimental autoimmune encephalomyelitis reveals that IL-10 mRNA expression correlates with recovery.

Experimental autoimmune encephalomyelitis (EAE) serves as an important animal model for understanding the events that lead to immune-mediated inflammation and tissue destruction within the central nervous system. We have utilized a murine adoptive transfer model of EAE and semiquantitative reverse transcriptase-polymerase chain reaction analysis to examine cytokine mRNA expression within the central nervous system in relation to the onset and resolution of paralysis associated with EAE. Spinal cord samples, obtained from mice as they progressed through discrete clinical stages of EAE, were examined for the expression of six cytokine genes (IL-1 alpha, IL-2, IL-4, IL-6, IL-10, and IFN-gamma). Distinct patterns of cytokine gene expression were observed during the acute, recovery, and chronic phases of EAE. The acute phase of disease was characterized by rapid increases in the levels of mRNA for IL-2, IL-4, IL-6, IFN-gamma, and IL-1 alpha. In fact, peak expression of several cytokine mRNA (e.g., IL-2, IL-4, IL-6, and IFN-gamma) occurred before the peak in clinical severity. In contrast, IL-1 alpha mRNA levels were elevated throughout the initial disease course. IL-10 mRNA demonstrated only modest increases during the acute phase of EAE. Stabilization of the clinical symptoms was characterized by rapid declines in the mRNA levels of IL-2, IL-4, IL-6, and IFN-gamma. The decreases in these four cytokine mRNA levels occurred concomitant with a dramatic rise in IL-10 mRNA. Finally, of the six cytokine mRNA examined, only IL-1 alpha, IFN-gamma, and IL-10 mRNA remained elevated during the early chronic stage. These results suggest that local cytokine production varies significantly during the course of EAE and that increases in discrete sets of cytokines are associated with the acute response and the recovery/chronic phase of disease.