Graft-versus-leukemia Effect of HLA-haploidentical Central-memory T-cells Expanded With Leukemic APCs and Modified With a Suicide Gene

Allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen (HLA)-haploidentical family donor (haplo-HSCT) is a readily available and potentially curative option for high-risk leukemia. In haplo-HSCT, alloreactivity plays a major role in the graft-versus-leukemia (GVL) effect, which, however, is frequently followed by relapse due to emerging leukemic cell variants that have lost the unshared HLA haplotype as a mechanism of immune escape. We report that stimulation of HLA-haploidentical donor T lymphocytes with leukemic antigen-presenting cells (L-APCs) expands a population of leukemia-reactive T cells, which, besides alloreactivity to unshared HLAs, contain leukemia-associated specificities restricted by shared HLAs. According to a preferential central-memory (T_CM) phenotype and to high interleukin (IL)-7Rα expression, these T cells persist in vivo and sustain a major GVL effect in a clinically relevant xenograft model. Moreover, we demonstrate that modifying L-APC–expanded T cells to express the herpes simplex virus thymidine kinase (HSV-tk) suicide gene enables their elimination with the prodrug ganciclovir (GCV), therefore providing a safety switch in case of graft-versus-host disease (GVHD). These results warrant the clinical investigation of L-APC–expanded T cells modified with a suicide gene in the setting of haplo-HSCT.     

Targeting the αv integrin/TGF-β axis improves natural killer cell function against glioblastoma stem cells

Glioblastoma multiforme (GBM), the most aggressive brain cancer, recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. We showed that GSCs, but not normal astrocytes, are sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. Mass cytometry and single-cell RNA sequencing of primary tumor samples revealed that GBM tumor–infiltrating NK cells acquired an altered phenotype associated with impaired lytic function relative to matched peripheral blood NK cells from patients with GBM or healthy donors. We attributed this immune evasion tactic to direct cell-to-cell contact between GSCs and NK cells via αv integrin–mediated TGF-β activation. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-β signaling or with TGFBR2 gene–edited allogeneic NK cells prevented GSC-induced NK cell dysfunction and tumor growth. These findings reveal an important mechanism of NK cell immune evasion by GSCs and suggest the αv integrin/TGF-β axis as a potentially useful therapeutic target in GBM.     

Monitoring of developing graft-versus-host disease mediated by herpes simplex virus thymidine kinase gene-transduced T cells

Introduction of the HSV-Tk suicide gene into allogeneic T cells offers the possibility to control developing host-reactive cells within the context of allogeneic bone marrow transplantation (BMT). Sensitive quantitative detection methods are a prerequisite to monitor genetically modified T cells in peripheral blood and tissues to study their involvement in graft-versus-host disease (GVHD)-induced lesions as well as their disappearance or persistence after ganciclovir (GCV)-induced suicide. We monitored the alloreactivity of HSV-Tk-transduced T cells after BMT by studying their in vivo distribution and quantity in peripheral blood and in tissues in a WAG/Rij into Brown Norway fully mismatched rat allogeneic BMT model. Genetically modified T cells were quantified in blood and tissues by fluorescence-activated cell sorting, immunohistochemical analysis, and real-time quantitative polymerase chain reaction (PCR) analysis. A significant increase in the number of allogeneic HSV-Tk(+) T cells was found in particular in spleen and lymph nodes and large numbers were found in tongue, skin, and intestines. In blood, an increase in HSV-Tk(+) T cells closely preceded clinical symptoms of GVHD. Real-time quantitative PCR proved to be a fast and accurate tool by which to quantify transduced T cells both in blood and tissues. This enables the study of the in vivo alloreactivity of retrovirus-transduced cells and the response of HSV-Tk-expressing T cells to GCV-induced suicide therapy. Furthermore, we showed the potential use to study specific cause-effect relationships in a broad range of animal and clinical studies involving genetically engineered cells.     

Influence of ex vivo expansion and retrovirus-mediated gene transfer on primary T lymphocyte phenotype and functions

To modulate alloreactivity after hematopoietic stem cell (HSC) transplantation, suicide gene-expressing donor T cells can be administered with an allogeneic T cell-depleted HSC graft. Immune competence of such cells is a critical issue. We have examined the impact of our ex vivo gene transfer protocol (12-day culture period including CD3/IL-2 activation, retrovirus-mediated gene transfer, and G418-based selection) on the phenotype and functional properties of gene-modified cells (GMC). GMC were compared with control cells that had been cultured in parallel with GMC, but nontransduced and nonselected, as well as with peripheral blood mononuclear cells (PBMC). Our data show that phenotypical modifications are similar in control cells and GMC, demonstrating that alterations result from the 12-day culture rather than from the transduction and/or selection process itself. Such modifications include a reversal of CD4/CD8 ratio, activated phenotype (increased expression of CD45RO, CD95, and HLA-DR), and acquisition or increased expression of co-stimulatory molecules (CD80, CD86, and CD40). This led to an enhanced allostimulating potential of GMC, as compared with resting T cells, when used as stimulating cells in mixed lymphocyte reactions. Conversely, when using them as responder cells in mixed lymphocyte reactions, GMC exhibited a rapid loss of alloreactivity that resulted both from culture-dependent and from transduction and/or selection-dependent events. In conclusion, the retrovirus-mediated gene transfer can be associated with major phenotypical and functional alterations that could have strong clinical implications (increased immunogenicity, reduced anti-leukemic effect). Thus, future T cell expansion protocols should try to improve not only cell expansion or gene transfer efficiency, but also T cell functions.     

An Inducible Caspase-9 Suicide Gene to Improve the Safety of Therapy Using Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSC) hold promise for regenerative therapies, though there are several safety concerns including the risk of oncogenic transformation or unwanted adverse effects associated with hiPSC or their differentiated progeny. Introduction of the inducible caspase-9 (iC9) suicide gene, which is activated by a specific chemical inducer of dimerization (CID), is one of the most appealing safety strategies for cell therapies and is currently being tested in multicenter clinical trials. Here, we show that the iC9 suicide gene with a human EF1α promoter can be introduced into hiPSC by lentiviral transduction. The transduced hiPSC maintain their pluripotency, including their capacity for unlimited self-renewal and the potential to differentiate into three germ layer tissues. Transduced hiPSC are eliminated within 24 hours of exposure to pharmacological levels of CID in vitro, with induction of apoptosis in 94–99% of the cells. Importantly, the iC9 suicide gene can eradicate tumors derived from hiPSC in vivo. In conclusion, we have developed a direct and efficient hiPSC killing system that provides a necessary safety mechanism for therapies using hiPSC. We believe that our iC9 suicide gene will be of value in clinical applications of hiPSC-based therapy.     

Nucleofection of DCs to Generate Multivirus-specific T Cells for Prevention or Treatment of Viral Infections in the Immunocompromised Host

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. To prevent and treat these, we have produced and infused cytotoxic T lymphocytes (CTLs) with specificity for Epstein–Barr virus (EBV), cytomegalovirus (CMV), and adenovirus (Adv), and shown that small numbers of infused cells proliferate in vivo and protect against all three viruses. Despite these encouraging results, broader implementation of this approach is limited by the need for infectious virus material (EBV), expensive production of clinical grade adenoviral vectors, and a prolonged (8–12 weeks) period of manufacture. There is also competition between virus-derived antigens within antigen-presenting cells (APCs), limiting extension to additional agents. We now describe an approach that uses DNA nucleofection of dendritic cells (DCs) with DNA plasmids that encode a range of immunodominant and subdominant viral antigens from CMV, EBV, BK, and Adv. Within 10 days, this methodology provides multivirus-reactive CTLs that lack alloreactivity. We further demonstrate that nucleofected DC stimulation can be combined with interferon-γ (IFN-γ) capture technology to produce even more rapid multivirus-CTL products for treatment of acute infection. These CTL generation procedures should increase the feasibility and applicability of T-cell therapy.     

Transient control of a virus-induced immunopathology by genetic immunosuppression

The ability to control T cell reactivity using suicide genes opens new perspectives for the treatment of T cell-mediated diseases. The therapeutic effect is achieved by the selective killing of thymidine kinase gene-modified activated T cells by ganciclovir (GCV). This strategy has been shown to control T cell alloreactivity efficiently after bone marrow or solid organ transplantation. Here, we aimed to determine whether an immunopathological process induced by a viral infection could be controlled by GCV when T cells express a thymidine kinase transgene. When transgenic mice were infected with the lymphocytic choriomeningitis virus, administration of GCV resulted in an efficient, but only transient, control of the immunopathological immune response. Further analysis revealed the existence of a minute population of GCV-insensitive T cells. These cells expand in response to the virus despite the presence of GCV and cause immunopathology before viral elimination is finally obtained. Thus, when confronted with a replicative virus, the efficacy of this genetic immunosuppression strategy is highly dependent on the presence of even small numbers of GCV-insensitive cells. These results emphasize the need for sufficient preclinical investigations with regard to the pathology and the nature of the immune response if suicide gene transfer is envisioned for new therapeutic indications.     

Reduced graft-versus-host disease-inducing capacity of T cells after activation, culturing, and magnetic cell sorting selection in an allogeneic bone marrow transplantation model in rats.

Graft-versus-host disease (GvHD), a major complication of allogeneic bone marrow transplantation, has been ascribed to mature T cells in the graft. Because T cells play an important role in engraftment of the bone marrow and decrease the probability of relapse of leukemia, a treatment strategy was developed to preserve the benefits of T cells in the graft and to control the severe complications of GvHD. This can be accomplished by the genetic modification of donor T cells with a suicide gene that allows their selective in vivo elimination and subsequently the abrogation of GvHD. For clinical benefit the alloreactivity of herpes simplex virus thymidine kinase (HSV-TK) gene-transduced T cells should be retained. Therefore, we investigated the influence of gene transduction and the selection procedure on T cells. We demonstrated that activation and culturing of T cells reduce their capacity to induce lethal GvHD in an allogeneic rat bone marrow transplantation model. Furthermore, positive immunomagnetic selection of gene-transduced T cells resulted in loss of the GvHD-inducing capacity of HSV-TK(+) T cells directly after MACS (magnetic cell sorting) selection; this loss could be recovered by a 1-day expansion of the selected T cells. No effect on alloreactivity was observed to be caused by the gene transduction procedure. Our study resulted in the development of an optimized culture and gene transduction protocol with preservation of T cell alloreactivity. Treatment of transplanted rats with ganciclovir resulted in a rapid reduction in the number of HSV-TK(+) T cells in the peripheral blood and in increased survival of the animals.     

[18F]FHBG PET/CT Imaging of CD34-TK75 Transduced Donor T Cells in Relapsed Allogeneic Stem Cell Transplant Patients: Safety and Feasibility

Described herein is a first-in-man attempt to both genetically modify T cells with an imagable suicide gene and track these transduced donor T cells in allogeneic stem cell transplantation recipients using noninvasive positron emission tomography/computerized tomography (PET/CT) imaging. A suicide gene encoding a human CD34-Herpes Simplex Virus-1-thymidine kinase (CD34-TK75) fusion enabled enrichment of retrovirally transduced T cells (TdT), control of graft-versus-host disease and imaging of TdT migration and expansion in vivo in mice and man. Analysis confirmed that CD34-TK75-enriched TdT contained no replication competent γ-retrovirus, were sensitive to ganciclovir, and displayed characteristic retroviral insertion sites (by targeted sequencing). Affinity-purified CD34-TK75⁺-selected donor T cells (1.0–13 × 10⁵)/kg were infused into eight patients who relapsed after allogeneic stem cell transplantation. Six patients also were administered 9-[4-(¹⁸F)fluoro-3-hydroxymethyl-butyl]guanine ([¹⁸F]FHBG) to specifically track the genetically modified donor T cells by PET/CT at several time points after infusion. All patients were assessed for graft-versus-host disease, response to ganciclovir, circulating TdT cells (using both quantitative polymerase chain reaction and [¹⁸F]FHBG PET/CT imaging), TdT cell clonal expansion, and immune response to the TdT. This phase 1 trial demonstrated that genetically modified T cells and [¹⁸F]FHBG can be safely infused in patients with relapsed hematologic malignancies after allogeneic stem cell transplantation.     

Methylguanine methyltransferase-mediated in vivo selection and chemoprotection of allogeneic stem cells in a large-animal model

Clinical application of gene therapy for genetic and malignant diseases has been limited by inefficient stem cell gene transfer. Here we studied in a clinically relevant canine model whether genetic chemoprotection mediated by a mutant of the DNA-repair enzyme methylguanine methyltransferase could circumvent this limitation. We hypothesized that genetic chemoprotection might also be used to enhance allogeneic stem cell transplantation, and thus we evaluated methylguanine methyltransferase-mediated chemoprotection in an allogeneic setting. We demonstrate that gene-modified allogeneic canine CD34+ cells can engraft even after low-dose total body irradiation conditioning. We also show that cytotoxic drug treatment produced a significant and sustained multilineage increase in gene-modified repopulating cells. Marking in granulocytes rose to levels of up to 98%, the highest in vivo marking reported to date to our knowledge in any large-animal or human study. Increases in transgene-expressing cells after in vivo selection provided protection from chemotherapy-induced myelosuppression, and proviral integration site analysis demonstrated the protection of multiple repopulating clones. Drug treatment also resulted in an increase in donor chimerism. These data demonstrate that durable, therapeutically relevant in vivo selection and chemoprotection of gene-modified cells can be achieved in a large-animal model and suggest that chemoprotection can also be used to enhance allogeneic stem cell transplantation.     

Differentiation and functional regulation of human fetal NK cells

The human fetal immune system is naturally exposed to maternal allogeneic cells, maternal antibodies, and pathogens. As such, it is faced with a considerable challenge with respect to the balance between immune reactivity and tolerance. Here, we show that fetal natural killer (NK) cells differentiate early in utero and are highly responsive to cytokines and antibody-mediated stimulation but respond poorly to HLA class I–negative target cells. Strikingly, expression of killer-cell immunoglobulin-like receptors (KIRs) did not educate fetal NK cells but rendered them hyporesponsive to target cells lacking HLA class I. In addition, fetal NK cells were highly susceptible to TGF-β–mediated suppression, and blocking of TGF-β signaling enhanced fetal NK cell responses to target cells. Our data demonstrate that KIR-mediated hyporesponsiveness and TGF-β–mediated suppression are major factors determining human fetal NK cell hyporesponsiveness to HLA class I–negative target cells and provide a potential mechanism for fetal-maternal tolerance in utero. Finally, our results provide a basis for understanding the role of fetal NK cells in pregnancy complications in which NK cells could be involved, for example, during in utero infections and anti-RhD–induced fetal anemia.     

Lentiviral vector conferring resistance to mycophenolate mofetil and sensitivity to ganciclovir for in vivo T-cell selection

Several clinical studies of gene-modified T cells have shown limited in vivo function of the cells, immunogenicity of the transgene, and lack of a selective advantage for gene-modified T cells. To address these problems, we developed a lentiviral vector (LV) that provides a selectable, proliferative advantage and potentially decreases immunogenicity for transduced T cells. The bicistronic vector expressed two genes linked with an internal ribosomal entry site. One gene is a variant of the inosine monophosphate dehydrogenase 2, inosine monophosphate dehydrogenase (IMPDH(IY)), conferring resistance to the immunosuppressive drug mycophenolate mofetil (MMF). The other is a suicide gene, herpes simplex virus thymidine kinase (HSV-TK), rendering proliferating cells sensitive to ablation with ganciclovir, fused to the selectable transmembrane marker DeltaCD34 (DeltaCD34/TK). Cells transduced with LV-DeltaCD34/TK.IMPDH(IY) were efficiently enriched by immunomagnetic selection for CD34, proliferated in 0.5-5 microM MMF, and were killed by 0.5-25 microg ml(-1) ganciclovir. We demonstrate efficient selection and killing of gene-modified cells and suggest LV-DeltaCD34/TK.IMPDH(IY)-transduced T cells could be used to facilitate allogeneic hematopoietic cell engraftment. The expression of IMPDH(IY) would allow in vivo selection with MMF, and DeltaCD34/TK expression would allow rapid and safe elimination of transduced T cells if graft-versus-host disease developed.     

Interleukin (IL)-1 promotes allogeneic T cell intimal infiltration and IL-17 production in a model of human artery rejection

Interleukin (IL) 1α produced by human endothelial cells (ECs), in response to tumor necrosis factor (TNF) or to co-culture with allogeneic T cells in a TNF-dependent manner, can augment the release of cytokines from alloreactive memory T cells in vitro. In a human–mouse chimeric model of artery allograft rejection, ECs lining the transplanted human arteries express IL-1α, and blocking IL-1 reduces the extent of human T cell infiltration into the artery intima and selectively inhibits IL-17 production by infiltrating T cells. In human skin grafts implanted on immunodeficient mice, administration of IL-17 is sufficient to induce mild inflammation. In cultured cells, IL-17 acts preferentially on vascular smooth muscle cells rather than ECs to enhance production of proinflammatory mediators, including IL-6, CXCL8, and CCL20. Neutralization of IL-17 does not reduce T cell infiltration into allogeneic human artery grafts, but markedly reduces IL-6, CXCL8, and CCL20 expression and selectively inhibits CCR6⁺ T cell accumulation in rejecting arteries. We conclude that graft-derived IL-1 can promote T cell intimal recruitment and IL-17 production during human artery allograft rejection, and suggest that targeting IL-1 in the perioperative transplant period may modulate host alloreactivity.     

Cell Fate Control Gene Therapy Based on Engineered Variants of Human Deoxycytidine Kinase

The safety of cell therapy applications can be enhanced by the introduction of Cell Fate Control (CFC) elements, which encode pharmacologically controlled cellular suicide switches. CFC Gene Therapy (CFCGT) offers the possibility of establishing control over gene-modified cells (GMCs) with regards to their proliferation, differentiation, or function. However, enzymes commonly employed in these approaches often possess poor kinetics and high immunogenicity. We describe a novel CFCGT system based on engineered variants of human deoxyCytidine Kinase (dCK) that overcomes limitations of current modalities. Mutants of dCK with rationally designed active sites that make them thymidine-activating were stably introduced into cells by recombinant lentiviral vectors (LVs). Transduced cells maintained growth kinetics and function. These dCK mutants efficiently activate bromovinyl-deoxyuridine (BVdU), L-deoxythymidine (LdT), and L-deoxyuridine (LdU), which are otherwise not toxic to wild-type cells. We show that mutant dCK-expressing Jurkat, Molt-4, and U87mg cells could be efficiently eliminated in vitro and in xenogeneic leukemia and tumor models in vivo. We also describe a fusion construct of the thymidine-activating dCK to the cytoplasmic tail-truncated LNGFR molecule and applications to in vivo eradication of primary human T cells. This novel CFCGT system offers unique plasticity with respect to the wide range of prodrugs it can potentiate, and can be used as a reliable safety switch in cell and gene therapy.     

Bone Marrow–generated Dendritic Cells Pulsed with Tumor Extracts or Tumor RNA Induce Antitumor Immunity against Central Nervous System Tumors

Recent studies have shown that the brain is not a barrier to successful active immunotherapy that uses gene-modified autologous tumor cell vaccines. In this study, we compared the efficacy of two types of vaccines for the treatment of tumors within the central nervous system (CNS): dendritic cell (DC)-based vaccines pulsed with either tumor extract or tumor RNA, and cytokine gene–modified tumor vaccines. Using the B16/F10 murine melanoma (B16) as a model for CNS tumor, we show that vaccination with bone marrow–generated DCs, pulsed with either B16 cell extract or B16 total RNA, can induce specific cytotoxic T lymphocytes against B16 tumor cells. Both types of DC vaccines were able to protect animals from tumors located in the CNS. DC-based vaccines also led to prolonged survival in mice with tumors placed before the initiation of vaccine therapy. The DC-based vaccines were at least as effective, if not more so, as vaccines containing B16 tumor cells in which the granulocytic macrophage colony-stimulating factor gene had been modified. These data support the use of DC-based vaccines for the treatment of patients with CNS tumors.     

Natural killer cell alloreactivity for leukemia therapy

Donor-versus-recipient natural killer (NK) cell alloreactivity derives from a mismatch between donor NK clones, carrying specific inhibitory receptors for self MHC class I molecules, and MHC class I ligands on recipient cells. When faced with mismatched allogeneic targets, these donor NK clones sense the missing expression of self HLA class I alleles and mediate alloreactions. Transplantation from NK alloreactive haploidentical donors controls acute myeloid leukemia relapse and improves engraftment without causing graft-versus-host disease.     

The immunology of experimental Chagas'' disease. 3. Rejection of allogeneic heart cells in vitro

Experiments that consisted of incubation of Trypanosoma cruzi-sensitized lymphocytes derived from chronically infected rabbits and from rabbits repeatedly immunized with a small particle or membrane fraction derived from homogenates of T. cruzi forms, showed destruction of allogeneic, parasitized and nonparasitized heart cells in vitro. Mononuclear cells collected from peripheral blood were incubated for 1 h at 37°C to isolate the lymphocytes. Following incubation, over 99% of the cells in the supernate were lymphocytes, which were utilized in these experiments. At the start of these experiments, 70–80% of the sensitized lymphocytes were unattached, small and round, with sparse filipodia. In the ensuing hours, marked heart cell destruction, similar to that seen in an active lesion when lymphocytes invade heart tissue, were observed. After 18 h incubation, about 65–70% of the lymphocytes were attached, larger, and rough surfaced. Inhibition of monocyte migration tests, each in the presence of the antigens of subcellular fractions of T. cruzi organisms and of allogeneic heart myofibers, indicated the presence of a cross-reacting antigen common to both the parasite and the heart in the small particle or membrane fractions. The particulate antigens of the 30,000 g, 35-min fraction of heart muscle gave rise to inhibition of monocyte migration as did the counterpart fraction derived from T. cruzi organisms. The destruction of nonparasitized target heart cells by T. cruzi-sensitized lymphocytes is an in vitro model of the chronic myocarditis of Chagas' disease, and the recognition of cross-reactive antigens of the host cell by T. cruzi-sensitized lymphocytes is believed to be the pathogenic basis for subsequent tissue injury in the chronic phase of this disease.     

Generation of T cells with lytic specificity for atypical antigens. III. Priming F1 animals with antigen-bearing cells also having reactivity for host alloantigens allows for potent lytic T cell responses

Here, we explore the conditions required for generating two different highly potent F1 antiparental killer cell populations to unusual antigens in rats. The first, L/DA anti-DA, has lytic specificity for two antigen systems: MTA, a mitochondrial antigen expressed on DA and DA Lewis (L) target cells restricted by RT1A class I molecules; and H, an antigen that maps to the class I-like RT1C region and is present only on parental target cells from donors homozygous at the major histocompatibility complex. The second killer population is generated in the reciprocal DA/L anti-DA combination and has lytic specificity only for the H antigen system. We show that the killer cells are T cells, and that generation of these F1 cytotoxic T lymphocytes (CTL) requires an in vivo priming step in which it is essential that the inoculated parental cells bear the relevant target antigens and possess alloreactivity for F1 host antigens. The requirement for alloreactivity and antigen on the same priming cell population suggests that these potent lytic responses depend on a situation akin to a hapten-carrier effect that bypasses otherwise ineffective helper responses by the host to these unusual antigens. Restimulation of F1 lymphocytes in culture is also necessary, requiring the presence of antigen on irradiated lymphoblast stimulator cells, but alloreactivity to responder cell antigens is not necessary; normal, nonactivated lymph node cells are completely ineffective as stimulators. For effective lysis, the target cells need not possess the potential for alloreactivity to responder F1 CTL. We also demonstrate in a preliminary way additional antigen systems defined by killer populations raised with other F1 antiparental strain combinations.     

The generation of killer cells to trinitrophenyl-modified allogeneic targets by lymphocyte populations negatively selected to strong alloantigens

Negatively selected mouse and rat lymphocyte populations, specifically deprived of alloreactivity to a particular major histocompatibility complex (MHC) haplotype, are nevertheless fully capable of responding to trinitrophenyl (TNP)-modified allogeneic stimulator cells and developing cytotoxic T-lymphocyte activity to TNP-altered allogeneic target cells. As for syngeneic systems, lytic expression of those responder killer cells also requires MHC identity between the target and stimulator cell populations. Such a finding argues strongly against two variations of the dual recognition hypothesis: like-like interactions and adaptive differentiation. Instead, these data favor either the altered self model or a third variation of the dual receptor model, where one of the relevent receptors is specific for the modifying antigen and the second is a low affinity receptor unable to be triggered in the absence of a modifying antigen.