Polyclonal Regulatory T Cell Therapy for Control of Inflammation in Kidney Transplants.

Early subclinical inflammation in kidney transplants is associated with later graft fibrosis and dysfunction. Regulatory T cells (Tregs) can reverse established inflammation in animal models. We conducted a pilot safety and feasibility trial of autologous Treg cell therapy in three kidney transplant recipients with subclinical inflammation noted on 6-month surveillance biopsies. Tregs were purified from peripheral blood and polyclonally expanded ex vivo using medium containing deuterated glucose to label the cells. All patients received a single infusion of ~320 × 106 (319, 321, and 363.8 × 106 ) expanded Tregs. Persistence of the infused Tregs was tracked. Graft inflammation was monitored with follow-up biopsies and urinary biomarkers. Nearly 1 × 109 (0.932, 0.956, 1.565 × 109 ) Tregs were successfully manufactured for each patient. There were no infusion reactions or serious therapy-related adverse events. The infused cells demonstrated patterns of persistence and stability similar to those observed in non-immunosuppressed subjects receiving the same dose of Tregs. Isolation and expansion of Tregs is feasible in kidney transplant patients on immunosuppression. Infusion of these cells was safe and well tolerated. Future trials will test the efficacy of polyclonal and donor alloantigen-reactive Tregs for the treatment of inflammation in kidney transplants.

Teplizumab treatment may improve C-peptide responses in participants with type 1 diabetes after the new-onset period: a randomised controlled trial.

AIMS/HYPOTHESIS: Type 1 diabetes results from a chronic autoimmune process continuing for years after presentation. We tested whether treatment with teplizumab (a Fc receptor non-binding anti-CD3 monoclonal antibody), after the new-onset period, affects the decline in C-peptide production in individuals with type 1 diabetes. METHODS: In a randomised placebo-controlled trial we treated 58 participants with type 1 diabetes for 4-12 months with teplizumab or placebo at four academic centres in the USA. A central randomisation centre used computer generated tables to allocate treatments. Investigators, patients, and caregivers were blinded to group assignment. The primary outcome was a comparison of C-peptide responses to a mixed meal after 1 year. We explored modification of treatment effects in subgroups of patients. RESULTS: Thirty-four and 29 subjects were randomized to the drug and placebo treated groups, respectively. Thirty-one and 27, respectively, were analysed. Although the primary outcome analysis showed a 21.7% higher C-peptide response in the teplizumab-treated group (0.45 vs 0.371; difference, 0.059 [95% CI 0.006, 0.115] nmol/l) (p = 0.03), when corrected for baseline imbalances in HbA(1c) levels, the C-peptide levels in the teplizumab-treated group were 17.7% higher (0.44 vs 0.378; difference, 0.049 [95% CI 0, 0.108] nmol/l, p = 0.09). A greater proportion of placebo-treated participants lost detectable C-peptide responses at 12 months (p = 0.03). The teplizumab group required less exogenous insulin (p < 0.001) but treatment differences in HbA(1c) levels were not observed. Teplizumab was well tolerated. A subgroup analysis showed that treatment benefits were larger in younger individuals and those with HbA(1c) <6.5% at entry. Clinical responders to teplizumab had an increase in circulating CD8 central memory cells 2 months after enrolment compared with non-responders. CONCLUSIONS/INTERPRETATIONS: This study suggests that deterioration in insulin secretion may be affected by immune therapy with teplizumab after the new-onset period but the magnitude of the effect is less than during the new-onset period. Our studies identify characteristics of patients most likely to respond to this immune therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT00378508 FUNDING: This work was supported by grants 2007-502, 2007-1059 and 2006-351 from the JDRF and grants R01 DK057846, P30 DK20495, UL1 RR024139, UL1RR025780, UL1 RR024131 and UL1 RR024134 from the NIH.

Clinical grade manufacturing of human alloantigen-reactive regulatory T cells for use in transplantation.

Regulatory T cell (Treg) therapy has the potential to induce transplantation tolerance so that immunosuppression and associated morbidity can be minimized. Alloantigen-reactive Tregs (arTregs) are more effective at preventing graft rejection than polyclonally expanded Tregs (PolyTregs) in murine models. We have developed a manufacturing process to expand human arTregs in short-term cultures using good manufacturing practice-compliant reagents. This process uses CD40L-activated allogeneic B cells to selectively expand arTregs followed by polyclonal restimulation to increase yield. Tregs expanded 100- to 1600-fold were highly alloantigen reactive and expressed the phenotype of stable Tregs. The alloantigen-expanded Tregs had a diverse TCR repertoire. They were more potent than PolyTregs in vitro and more effective at controlling allograft injuries in vivo in a humanized mouse model.

The Extra-Islet Pancreas Supports Autoimmunity in Human Type 1 Diabetes.

In autoimmune Type 1 diabetes (T1D), immune cells progressively infiltrate and destroy the islets of Langerhans - islands of endocrine tissue dispersed throughout the pancreas. However, it is unclear how this process, called 'insulitis', develops and progresses within this organ. Here, using highly multiplexed CO-Detection by indEXing (CODEX) tissue imaging and cadaveric pancreas samples from pre-T1D, T1D, and non-T1D donors, we examine pseudotemporal-spatial patterns of insulitis and exocrine inflammation within large pancreatic tissue sections. We identify four sub-states of insulitis characterized by CD8 + T cells at different stages of activation. We further find that exocrine compartments of pancreatic lobules affected by insulitis have distinct cellularity, suggesting that extra-islet factors may make particular lobules permissive to disease. Finally, we identify "staging areas" - immature tertiary lymphoid structures away from islets where CD8 + T cells appear to assemble before they navigate to islets. Together, these data implicate the extra-islet pancreas in autoimmune insulitis, greatly expanding the boundaries of T1D pathogenesis.

Cloned cytotoxic T lymphocytes that recognize an I-A region product in the context of a class I antigen.

Cloned CTLs QM3 and QM7 isolated from a bulk CTL line B10.QBR anti-B10.MBR recognized a combination of the H-2Kb molecule and an I-Ak subregion gene product. Such a combinatorial specificity was revealed by complementation of the target antigen in F1 animals between two negative parental strains carrying H-2Kb and I-Ak, respectively. We confirmed the involvement of the H-2Kb molecule by blocking killing with anti-Kb mAb and failure of certain mutant H-2Kb genes to complement with I-Ak to generate the determinant in F1 animals. Although the nature of the I-Ak subregion gene product is not definitive, there was a correlation between the expression of Ia antigens on the cell surface and susceptibility of the cells to lysis by these CTLs, suggesting that it is the classical I-Ak class II antigen.

Dissociation of intracellular signaling pathways in response to partial agonist ligands of the T cell receptor.

The T cell receptor (TCR) is a versatile receptor able to generate different signals that result in distinct T cell responses. The pattern of early signals is determined by the TCR binding kinetics that control the ability of the ligand to coengage TCR and coreceptor. Coengagement of TCR and CD4 results in an agonist signaling pattern with complete tyrosine phosphorylation of TCR subunits, and recruitment and activation of ZAP-70. In contrast, TCR engagement without CD4 coengagement causes a partial agonist type of signaling, characterized by distinct phosphorylation of TCR subunits and recruitment but no activation of ZAP-70. The pathways triggered by partial agonist signaling are unknown. Here, we show that agonists cause association of active lck and active ZAP-70 with p120-GTPase-activating protein (p120-GAP). These associations follow engagement of CD4 or CD3, respectively. In contrast, partial agonists do not activate lck or ZAP-70, but induce association of p120-GAP with inactive ZAP-70. Despite these differences, both agonist and partial agonist signals activate the mitogen-activated protein kinase (MAPK) pathway. However, MAPK activation by partial agonists is transient, supporting a kinetic, CD4-dependent model for the mechanism of action of variant TCR ligands. Transient MAPK activation may explain some of the responses to TCR partial agonists and antagonists.

Evidence for a regulatory idiotypic network in the in vivo response to H-2 antigens.

Treatment of BALB/c mice with purified pig antiidiotype to 11-4.1 (anti-H-2Kk) monoclonal antibody has been found previously to induce the appearance of idiotype-bearing molecules (Id') in the serum of these mice, in the absence of detectable antigen-binding activity. In the present study we examined the effect of subsequent immunization of such antiidiotype-primed mice with the original H-2Kk antigen. Skin grafting of virgin BALB/c mice with BALB.K skin did not generate any detectable Id' antibodies when tested by enzyme-linked immunosorbent assay (ELISA). In contrast, grafting of antiidiotype-primed mice with BALB.K skin specifically boosted ther serum level of Id' molecules. Challenge of antiidiotype-primed mice with either B10.D2 or rat skin had no effect on the production of such Id' molecules. Absorption studies demonstrated that the majority of Id' molecules induced by H-2Kk antigenic stimulus and detected in ELISA are antigen-nonbinding molecules, thus indicating specific restimulation by the original H-2Kk antigen of nonbinding idiotype-positive B cell clones. The relevance of these findings to the existence of network interactions in the immune response to H-2 antigens is discussed.

Antiidiotypes against anti-H-2 monoclonal antibodies. V. In vivo antiidiotype treatment induces idiotype-specific helper T cells.

Mice have been treated in vivo with xenogeneic antiidiotypes prepared against a murine monoclonal anti-H-2Kk antibody, 11-4.1. B cell immune responses have been found to be altered by such treatment as evidenced by a modification in the idiotypic repertoire of the humoral response to H-2 antigens. Transfer of purified T cells into nude mice before anti-idiotype treatment showed that T cells are involved in the induction of idiotope-bearing antibodies by xenogeneic antiidiotype. Studies using bone marrow chimeras indicate that the environment in which either T or B cells mature does not appear to alter VH region genetic control of induction of antiidiotype-induced molecules. By adoptive transfer studies, T cells from antiidiotype-treated mice were found capable of modifying the idiotypic repertoire of B cells subsequently exposed to antigen even when the T cells were obtained from antiidiotype-primed mice of inappropriate allotype. Although it still must be determined whether idiotypic or antiidiotypic T cells are involved in such B cell idiotype regulation, these results indicate that some T cell functions are altered by xenogeneic antiidiotypes prepared against B cell products and suggest that T cell immunity to major histocompatibility complex antigens may also be affected by such reagents.

Tolerance of T cell receptor gamma/delta cells in the intestine.

The present study examined the mechanism(s) of tolerance induction for intestinal intraepithelial lymphocytes (iIELs) using an alloantigen (Ag)-specific gamma/delta T cell receptor (TCR gamma/delta) transgenic (Tg) model. In Tg Ag-bearing H-2b/d mice (Tgb/d), Tg iIELs were Thy-1-, CD44+, CD45R (B220)+, and CD5+, whereas in syngeneic Tgd/d mice, iIELs were Thy-1+, CD44-, and CD45R- with a subset of CD5+ cells. Previously, we had shown that tolerance for Tgb/d iIELs involved functional anergy and deletion (Barrett, T. A., M. L. Delvy, D. M. Kennedy, L. Lefrancois, L. A. Matis, A. L. Dent, S. M. Hedrick, and J. A. Bluestone. 1992. J. Exp. Med. 175:65). In this study we demonstrate that Tgb/d iIELs expressing dull levels of Thy-1 proliferated in the presence of exogenous rIL-2. A direct precursor-product relationship between the Thy-1+-responsive iIELs and the tolerant Thy-1dul/- iIELs was demonstrated by adoptive transfer into severe combined immunodeficient (SCID) mice. Tg Thy-1+ iIELs reconstituting Ag+ but not Ag- SCID mice downregulated Thy-1 after Ag exposure in vivo. Analysis of bone marrow (BM) chimeras demonstrated the persistence of Tg IELs in all Ag+ chimeras although a modest degree of clonal deletion was apparent. The greatest percentage of Tg IELs were detected when Ag was restricted to radioresistant cells (e.g., epithelial cells) compared with BM-derived antigen-presenting cells (APC). This was especially apparent in thymectomized chimeric mice. Consistent with the notion that Ag-bearing epithelial cells may be poor APC, isolated intestinal epithelial cells from Ag-bearing mice failed to stimulate Tg iIELs compared with splenic APC. These studies suggest that the major population of TCR gamma/delta iIELs were probably extrathymically derived and encountered self-Ag on intestinal epithelial cells. The induction of tolerance likely involved an activation event resulting in downregulation of Thy-1. These mechanisms of tolerance for TCR gamma/delta iIELs led to the persistence of a reservoir of self-reactive T cells with the potential for mediating autoimmune disease.

Idiotypes of anti-Ia antibodies. I. Expression of the 14-4-4S idiotype in humoral immune responses.

The idiotype of a mouse monoclonal anti-I-E antibody, 14-4-4S, has been studied using a heterologous anti-idiotypic reagent. This antibody recognizes Ia. 7, an antigenic specificity present in all strains expressing a product of the I-E subregion. Expression of the 14-4-4S idiotype in humoral immune responses was analyzed by an idiotype-specific enzyme-linked immunosorbent assay system. The idiotype was readily detectable in C3H.SW anti-C3H alloantisera, the same immunization combination from which the hybridoma was derived. Absorption analysis demonstrated the anti-I-E specificity of the idiotype-positive molecules in these alloantisera. Penetrance of idiotype expression was high among individual C3H.SW immune mice (9 of 10 tested). To examine genetic requirements for idiotype expression, an immunization was performed using as responders CWB mice, congenic with C3H.SW but differing at the heavy chain allotype loci. Immune sera of individual CWB mice contained very little or no idiotype, demonstrating that levels of idiotype expression are influenced by allotype-linked genes, although the influence of other genes has not been ruled. The 14-4-4S idiotype therefore represents a shared idiotype of anti-Ia antibodies and provides opportunities for analysis of the idiotypes of cellular receptors for the corresponding Ia antigen.

Alloresistance to engraftment of allogeneic donor bone marrow is mediated by an Lyt-2+ T cell in mixed allogeneic reconstitution (C57BL/10Sn + B10.D2/nSn----C57BL/10Sn).

In the mixed allogeneic reconstitution (B10 + B10.D2----B10) model, alloresistance to engraftment of allogeneic donor results if the syngeneic component of the mixed bone marrow inoculum is not depleted of Lyt-2+ cells before transplantation. Resultant experimental animals repopulate as fully syngeneic, reject B10.D2 skin allografts, and are reactive to B10.D2 lymphoid cells in vitro, as assessed by mixed lymphocyte culture proliferative and cellular cytotoxicity assays. In contrast, depletion of Lyt-2-reactive cells from the syngeneic component of the mixed bone marrow inoculum results in mixed lymphopoietic chimerism and specific in vivo transplantation tolerance to B10.D2 allogeneic donor skin grafts and in vitro unreactivity to B10.D2 lymphoid elements. Full reactivity to third party is evident both in vitro and in vivo in these animals. This model may be helpful in further study of the syngeneic host-type cell phenotypes responsible for alloresistance to bone marrow engraftment.

Immune deviation of 2C transgenic intraepithelial lymphocytes in antigen-bearing hosts.

The present study examined self-tolerance for T cell receptor (TCR) alpha beta intestinal intraepithelial lymphocytes (iIELs) using the 2C transgenic (Tg) mouse model specific for a peptide antigen (Ag) presented by the class I major histocompatibility complex H-2Ld. Although Tg+ T cells were largely deleted from the periphery of Ag+ mice, equivalent numbers of Tg iIELs were present in Ag+ compared to Ag- mice. Tg iIELs in Ag- mice contained CD8 alpha beta, CD8 alpha alpha, and CD4-CD8- subsets, whereas only CD8 alpha alpha and CD4-CD8- Tg iIEL subsets were detected in Ag+ mice. Analysis of surface markers revealed that Tg iIELs in Ag+ mice expressed decreased levels of Thy-1 and increased CD45R/B220 as compared to Ag- Tg iIELs. In response to activation with exogenous peptide or immobilized anti-TCR mAB, iIELs from Ag- mice proliferated at high levels and produced interleukin (IL)-2 and interferon (IFN)-gamma, while Tg+ iIELs from Ag+ mice proliferated at low levels and failed to produce detectable IL-2 or IFN-gamma. Activation of sorted iIEL subsets from Ag- mice revealed that CD8 alpha alpha and CD4-CD8- subsets produced low levels of IL-2 and IFN-gamma in response to activation with antigen-presenting cells and added peptide or immobilized anti-TCR mAb, while CD8 alpha beta + iIELs responded to endogenous levels of peptide. In response to APC and exogenous peptide, sorted iIEL subsets from Ag+ mice produced IL-2 and IFN-gamma, and proliferated at greatly reduced levels compared to corresponding subsets from Ag- mice. Analysis of cytokine mRNA levels revealed that activation in vitro induced IL-2 mRNA only in Ag-, but not Ag+ iIELs, whereas a high level of IL-4 mRNA induction was detected in Tg+ iIELs from Ag+ mice, and to a lesser degree, from Ag- mice. These data suggest that tolerance for Tg+ iIELs resulted in the deletion of CD8 alpha beta + subsets and the persistence of Tg+ iIEL subsets with decreased sensitivity to endogenous levels of self-peptide. A comparison of the cytokine profiles expressed by Tg+ iIEL subsets in Ag- and Ag+ mice suggested that tolerance induction had involved the functional deviation of cells from TC1 (T helper-1-like) to a less inflammatory TC2 (T helper-2-like) phenotype capable of mediating humoral immune responses in the mucosa.

Peptide-induced conformational changes in class I heavy chains alter major histocompatibility complex recognition.

Small peptides, derived from endogenous proteins bind within the antigen binding groove created by the beta-pleated sheets and alpha helices of the alpha 1 and alpha 2 domains of the class I molecule of the major histocompatibility complex (MHC). However, the precise role of peptide in class I MHC conformation remains unclear. Here, we have shown that, in at least some instances, changes induced in the MHC molecule by the binding of distinct peptides can be identified as specific alterations in serological epitopes expressed on the class I protein. The nature of specific peptides expressed by class I-bearing cells may, therefore, have a dramatic influence on T cell development, self-tolerance, and alloreactivity.

CTLA-4 ligation blocks CD28-dependent T cell activation.

CTLA-4 is a CD28 homologue believed to be a negative regulator of T cell function. However, the mechanism of this downregulatory activity is not well understood. The present study was designed to examine the effect of CTLA-4 ligation on cytokine production, cell survival, and cell cycle progression. The results demonstrate that the primary effect of CTLA-4 ligation is not the induction of apoptosis. Instead, CTLA-4 signaling blocks IL-2 production, IL-2 receptor expression, and cell cycle progression of activated T cells. Moreover, the effect of CTLA-4 signaling was manifested after initial T cell activation. Inhibition of IL-2 receptor expression and cell cycle progression was more pronounced at late (72 h) time points after initial activation. The effects of anti-CTLA-4 mAbs were most apparent in the presence of optimal CD28-mediated costimulation consistent with the finding that CTLA-4 upregulation was CD28-dependent. Finally, the addition of exogenous IL-2 to the cultures restored IL-2 receptor expression and T cell proliferation. These results suggest that CTLA-4 signaling does not regulate cell survival or responsiveness to IL-2, but does inhibit CD28-dependent IL-2 production.

A single amino acid substitution in the alpha 3 domain of an H-2 class I molecule abrogates reactivity with CTL.

We previously described a somatic cell expressing a variant H-2Dd molecule that did not serve as a target for alloreactive anti-Dd CTL. The mutant cell line had been isolated by its failure to express a serological epitope present on the H-2Dd alpha 3 domain. In the present study the alpha 3 domain of the Dd molecule of this somatic cell variant was sequenced and a single nucleotide change resulting in a glutamic acid to lysine substitution at residue 227 was identified. This change was reproduced in the cloned H-2Dd gene by oligonucleotide-directed mutagenesis. Cells transfected with this mutant gene were not killed by anti-H-2Dd CTL. Because previous studies using hybrid H-2 class I molecules had established that the alpha 3 domain does not express allele-specific determinants recognized by CTL, our results raise the possibility that residues in the alpha 3 domain of H-2 class I molecules are critical for CTL recognition and constitute a conserved (or monomorphic) determinant recognized by CTL.

Junctional sequences influence the specificity of gamma/delta T cell receptors.

T lymphocytes bearing the gamma/delta T cell receptor (TCR-gamma/delta) express a limited number of germline variable gene segments, generating receptor sequence diversity primarily through junctional mechanisms. To examine the role of V(D)J junctional sequences in antigen recognition by TCR-gamma/delta, we derived an alloreactive murine TCR-gamma/delta+ T cell line, LKD1, specific for the I-Ad class II major histocompatibility complex (MHC) molecule, and compared its receptor with that expressed by a previously characterized class II MHC alloreactive T cell line, LBK5, specific for I-Ek,b,s Ia molecules. Both LKD1 and LBK5 express receptors encoded by rearranged V gamma 1.2J gamma 2 and V delta 5D delta 2J delta 1 gene elements, differing in sequence only in the V(D)J junctional regions of the gamma and delta genes. These results demonstrate that junctionally encoded sequences corresponding to the putative third complementarity determining region can influence the antigen specificity of TCR-gamma/delta.

T cell receptor-gamma/delta cells protect mice from herpes simplex virus type 1-induced lethal encephalitis.

Increased numbers of T cell receptor (TCR)-gamma/delta cells have been observed in animal models of influenza and sendai virus infections, as well as in patients infected with human immunodeficiency virus and herpes simplex virus type 1 (HSV-1). However, a direct role for TCR-gamma/delta cells in protective immunity for pathogenic viral infection has not been demonstrated. To define the role of TCR-gamma/delta cells in anti-HSV-1 immunity, TCR-alpha-/- mice treated with anti- TCR-gamma/delta monoclonal antibodies or TCR-gamma/delta x TCR-alpha/beta double-deficient mice were infected with HSV-1 by footpad or ocular routes of infection. In both models of HSV-1 infection, TCR-gamma/delta cells limited severe HSV-1-induced epithelial lesions and greatly reduced mortality by preventing the development of lethal viral encephalitis. The observed protection resulted from TCR-gamma/delta cell-mediated arrest of both viral replication and neurovirulence. The demonstration that TCR-gamma/delta cells play an important protective role in murine HSV-1 infections supports their potential contribution to the immune responses in human HSV-1 infection. Thus, this study demonstrates that TCR-gamma/delta cells may play an important regulatory role in human HSV-1 infections.

CTLA-4: a negative regulator of autoimmune disease.

CTLA-4, a CD28 homologue expressed on activated T cells, binds with high affinity to the CD28 ligands, B7-1 (CD80) and B7-2 (CD86). This study was designed to examine the role of CTLA-4 in regulating autoimmune disease. Murine relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE) is a demyelinating disease mediated by PLP139-151-specific CD4+ T cells in SJL/J mice. Anti-CTLA-4 mAbs (or their F(ab) fragments) enhanced in vitro proliferation and pro-inflammatory cytokine production by PLP139-151-primed lymph node cells. Addition of either reagent to in vitro activation cultures potentiated the ability of T cells to adoptively transfer disease to naive recipients. In vivo administration of anti-CTLA-4 mAb to recipients of PLP139-151-specific T cells resulted in accelerated and exacerbated disease. Finally, anti-CTLA-4 treatment of mice during disease remission resulted in the exacerbation of relapses. Collectively, these results suggest that CTLA-4 mediates the downregulation of ongoing immune responses and plays a major role in regulating autoimmunity.

Nonmitogenic anti-CD3 monoclonal antibodies deliver a partial T cell receptor signal and induce clonal anergy.

Anti-CD3 monoclonal antibodies (mAbs) are potent immunosuppressive agents used in clinical transplantation. However, the activation-related adverse side effects associated with these mAbs have prompted the development of less toxic nonmitogenic anti-CD3 mAb therapies. At present, the functional and biochemical consequences of T cell exposure to nonmitogenic anti-CD3 is unclear. In this study, we have examined the early signaling events triggered by a nonmitogenic anti-CD3 mAb. Like the mitogenic anti-CD3 mAb, nonnmitogenic anti-CD3 triggered changes in the T cell receptor (TCR) complex, including zeta chain tyrosine phosphorylation and ZAP-70 association. However, unlike the mitogenic anti-CD3 stimulation, nonmitogenic anti-CD3 was ineffective at inducing the highly phosphorylated form of zeta (p23) and tyrosine phosphorylation of the associated ZAP-70 tyrosine kinase. This proximal signaling deficiency correlated with minimal phospholipase Cgamma-1 phosphorylation and failure to mobilize detectable Ca2+. Not only did biochemical signals delivered by nonmitogenic anti-CD3 resemble altered peptide ligand signaling, but exposure of Th1 clones to nonmitogenic anti-CD3 also resulted in functional anergy. Finally, a bispecific anti-CD3 X anti-CD4 F(ab)'2 reconstituted early signal transduction events and induced proliferation, suggesting that defective association of lck with the TCR complex may underlie the observed signaling differences between the mitogenic and nonmitogenic anti-CD3.

The efficiency of CD4 recruitment to ligand-engaged TCR controls the agonist/partial agonist properties of peptide-MHC molecule ligands.

One hypothesis seeking to explain the signaling and biological properties of T cell receptor for antigen (TCR) partial agonists and antagonists is the coreceptor density/kinetic model, which proposes that the pharmacologic behavior of a TCR ligand is largely determined by the relative rates of (a) dissociation ofligand from an engaged TCR and (b) recruitment oflck-linked coreceptors to this ligand-engaged receptor. Using several approaches to prevent or reduce the association of CD4 with occupied TCR, we demonstrate that consistent with this hypothesis, the biological and biochemical consequence of limiting this interaction is to convert typical agonists into partial agonist stimuli. Thus, adding anti-CD4 antibody to T cells recognizing a wild-type peptide-MHC class II ligand leads to disproportionate inhibition of interleukin-2 (IL-2) relative to IL-3 production, the same pattern seen using a TCR partial agonist/antagonist. In addition, T cells exposed to wild-type ligand in the presence of anti-CD4 antibodies show a pattern of TCR signaling resembling that seen using partial agonists, with predominant accumulation of the p21 tyrosine-phosphorylated form of TCR-zeta, reduced tyrosine phosphorylation of CD3epsilon, and no detectable phosphorylation of ZAP-70. Similar results are obtained when the wild-type ligand is presented by mutant class II MHC molecules unable to bind CD4. Likewise, antibody coligation of CD3 and CD4 results in an agonist-like phosphorylation pattern, whereas bivalent engagement of CD3 alone gives a partial agonist-like pattern. Finally, in accord with data showing that partial agonists often induce T cell anergy, CD4 blockade during antigen exposure renders cloned T cells unable to produce IL-2 upon restimulation. These results demonstrate that the biochemical and functional responses to variant TCR ligands with partial agonist properties can be largely reproduced by inhibiting recruitment of CD4 to a TCR binding a wild-type ligand, consistent with the idea that the relative rates of TCR-ligand disengagement and of association of engaged TCR with CD4 may play a key role in determining the pharmacologic properties of peptide-MHC molecule ligands. Beyond this insight into signaling through the TCR, these results have implications for models of thymocyte selection and the use of anti-coreceptor antibodies in vivo for the establishment ofimmunological tolerance.