Adaptive tolerance of CD4+ T cells in vivo: multiple thresholds in response to a constant level of antigen presentation.

The in vivo T cell response to persistent Ag contains a hyporesponsive phase following an initial expansion and subsequent partial deletion of the responding cells. The mechanism(s) responsible for this tolerance process is poorly understood. In this study, we describe a new paired transgenic model (TCR and Ag), which within 7-14 days produces 20-40 million hyporesponsive T cells. This state is characterized by an 85-95% reduction in all cytokine production, an impairment of re-expression of CD25 and CD69, and a desensitization of the proliferative response to Ag. TCR levels were normal, and in vivo mixing experiments showed no evidence for active suppression. The hyporesponsiveness partially dissipated without proliferation when the cells were transferred into a non-Ag-bearing host. If the second host expressed Ag, the T cells initially regained responsiveness, but then slowly entered an even deeper state of tolerance characterized by an additional 7- to 10-fold lowering of cytokine production and a greater desensitization of proliferation. Surprisingly, this readaptation took place with the same level of Ag presentation, suggesting that other parameters can influence the tolerance threshold. Both the readjustment in sensitivity and the reversal without Ag convincingly demonstrate for the first time a truly adaptive tolerance process in CD4+ T cells in vivo.

Biallelic expression of the IL-2 locus under optimal stimulation conditions.

Recent experiments have suggested that the IL-2 locus is monoallelically expressed. We tested this hypothesis using TCR-transgenic mice carrying one inactivated IL-2 allele. The frequency in single-cell assays of IL-2-producing cells following optimal stimulation by antigen and antigen-presenting cells was equivalent to that from wild-type mice, but the amount of IL-2 produced per cell was twofold less. Similar observations were made by intracellular staining for IL-2, although stimulation in bulk culture was less optimal, showing only a 1.7-fold difference. Importantly, the frequency of responding cells from the heterozygotes was less than from the wild-type mice if the IL-2 assay was performed after only 24 - 30 h of activation, suggesting that the targeted allele could compete with the normal allele early after stimulation and give the misimpression that the heterozygotes had fewer IL-2-producing cells. These data strongly argue that the IL-2 locus can be expressed biallelically under optimum stimulation conditions.

Induction of competence to respond to IL-4 by CD4+ T helper type 1 cells requires costimulation.

Rested murine CD4+ Th1 clones do not produce IL-4, but have previously been shown to be capable of responding to IL-4 if they are first activated with Ag and APC. In this study, we have examined the activation requirements for induction of competence to respond to IL-4 in these clones. TCR occupancy alone (given either as chemically fixed APC and Ag, anti-CD3, Con A, or ionomycin and PMA) was inadequate, but the addition of a source of costimulation to any of these stimuli resulted in complete induction of competence to respond to IL-4. Pretreatment of the Th1 clones with TCR occupancy alone induced an anergic state from which subsequent full stimulation with Ag and APC failed to give IL-4 responsiveness. Pretreatment of the cells with IL-2 alone was an inadequate signal to induce IL-4 responsiveness and only a partial response was obtained when TCR occupancy was combined with IL-2. Addition of anti-IL-2 and anti-IL-2R antibodies during full activation with APC and Ag gave a 50% inhibition of competence induction. These results demonstrate that costimulation, in addition to its role in IL-2 production, is an important second signal for inducing T cells to become competent to respond to IL-4.

Clonal anergy blocks the response to IL-4, as well as the production of IL-2, in dual-producing T helper cell clones.

In this report we extend the in vitro clonal anergy model to examine the regulation of proliferation in T cells that secrete both IL-2 and IL-4. Newly cloned Ag-specific murine T cells are shown to depend on both IL-2 and IL-4 synthesis for maximal proliferation. Whereas IL-2 responsiveness is constitutive in these cells, IL-4 responsiveness develops only after Ag and APC stimulation. Remarkably, proliferation of these cells to Ag is sensitive to inhibition by clonal anergy, even though IL-4 synthesis remains inducible. Anergy in these cells is associated with an inability to respond to IL-4, in addition to the development of an IL-2 production defect. The results suggest that anergy induction may be capable of preventing the clonal expansion of autoreactive T cells producing both IL-2 and IL-4 in vivo.

An intracellular calcium increase and protein kinase C activation fail to initiate T cell proliferation in the absence of a costimulatory signal.

Resting T lymphocytes proliferate in response to a combination of a calcium ionophore and a phorbol ester. This observation suggests that an increase in intracellular calcium free ion concentration [Ca2+]i and activation of protein kinase C (PKC) are sufficient signaling events for the initiation of T cell proliferation. In contrast, an accessory cell-generated costimulatory signal, acting independently of the rise in [Ca2+]i and PKC activation, is required for Ag-induced proliferation of type I T cell clones. We now report that this costimulatory signal is unexpectedly also being delivered via a cell-cell interaction during the response to ionomycin and phorbol ester. In the absence of this signal (at limiting cell numbers), T cells fail to divide. We also demonstrate that proliferation in response to immobilized anti-CD3 mAb requires the cell-cell interaction. These results suggest a model of T cell stimulation in which activation of a costimulatory signaling pathway is important in the regulation of the IL-2 gene, and only in the presence of this (third) signal can an increase in [Ca2+]i and PKC activity induce T cell proliferation. Such a model predicts that IL-2-dependent expansion of T cell clones in vivo in response to Ag receptor occupancy requires the delivery of an independent accessory cell-derived co-stimulatory signal.

Scale-up methodology for the preparative purification of peptide M.

An octadecapeptide, peptide M, the epitope of a retinal protein that induces experimental autoimmune uveitis, was synthesized and purified by preparative reversed-phase chromatography. The flow-rate and gradient conditions for maximum separation of impurities were determined on a 30 x 0.39 cm I.D. column of Delta Pak (15-microns spherical C18-bonded silica with 300-A pores). The maximum amount of peptide that was resolved under these conditions was then determined experimentally. Using a scale factor dependent on the square of the column diameters, the flow-rate and amount loaded were increased 164 times on a 30 x 5 cm I.D. column of the same packing. The same resolution was achieved. Batches of 200-342 mg were chromatographed with reproducible results, providing a total yield of 394 mg of pure peptide.