| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Receptor-Targeted Antigen Loading in Conjunction with Double-Stranded RNA-Mediated Immune Modulation
* Alliance Pharmaceuticals and
MultiCell Immunotherapeutics, San Diego, CA 92121;
Mount Sinai School of Medicine, New York, NY 11029; and
University of Missouri, Columbia, MO 65212
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
R, but kept in check by ITIM motif (ITIM+) FcRIIB-mediated mechanisms. In addition to interfering with the functionality of ITIM+ FcR, effective anti-idiotypic and antitumoral immunity can be achieved by FcR-targeted delivery of epitope in conjunction with administration of stimulatory motifs such as dsRNA, correcting the ineffective response to idiotypic epitopes. The immune process initiated by FcR-mediated targeting of epitope together with dsRNA, resulted in control of tumor growth, establishment of immune memory and protection against tumors bearing antigenic variants. In summary, targeted delivery of MHC class I-restricted epitopes via ITAM+ FcR, in conjunction with use of TLR-binding immune stimulatory motifs such as dsRNA, overcomes suboptimal responses to idiotypic determinants and may constitute a novel approach for the treatment of a broad range of malignancies. Finally, the results shed light on the mechanisms regulating the idiotypic network and managing the diversity associated with immune receptors. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
Previous reports show that malignancies caused by oncoviruses are associated with continuous expression of non-self TAA (10, 11, 12), ruling out Ag loss as universal mechanism of immune escape. Furthermore, despite the quasi-intact T cell repertoire, previous studies have suggested that in the case of TAA that are of non-self origin, a specific T cell response is present but inadequate, for example dominated by T2 cells (13, 14), and unable to clear tumoral cells. Thus, immune deviation rather than Ag-loss or tolerance, may be a causative factor for the lack of control of tumors expressing non-self or Ags.
A particular case is represented by lymphoid malignancies that express neo-self Ags in the form of Id borne by TCR or Ig receptors. It is not clear to what extent such determinants are recognized or what the nature of immune response is during the progression of Id+ lymphoid malignancies. For example, despite the neo-self nature of such determinants, the immune response against Id expressed by malignant cells may occur (15, 16, 17) but in a suboptimal fashion, in particular in later stage disease, resulting in a failure of the endogenous defense mechanisms to control the tumoral process. In support of this possibility, clinical studies conducted over the last decade using Id in combination with KLH or GM-CSF, or infusion of Id-pulsed immature dendritic cells (DC), showed that although numerous patients mounted anti-Id immunity (i.e., Ab, Th, and/or CTL), the magnitude was generally reduced, the immune profile dominated by T2 cells and the clinical impact relatively modest (17, 18), somewhat moderating the optimism generated by earlier data (19). In general, despite the acknowledged capability of CTL immune responses to remove cells that express new Ags and the documented circumstantial evidence on generation of anti-Id cytotoxic immunity in mice (20, 21) and humans (22), it is not known whether neo-self MHC class I-restricted Id generated stochastically can play a significant role in the immune-mediated containment of malignant or nonmalignant lymphoid proliferations.
From a different point of view, Igs have been used as carriers for B and T cell epitopes, for the purpose of induction of prophylactic and therapeutic responses in preclinical models (23, 24). That method has been largely promoted by the fact that peptides have a rather poor immune activity, due to their suboptimal pharmacokinetic profile. The alternative, ex vivo peptide loading of APC, implies individualized therapies and thus, it is more difficult to implement therapeutically on a large scale. It has been shown that recombinant Ig, carrying an MHC class II-restricted epitope derived from influenza virus within CDR3 of the H chain, effectively targets DC via FcR, resulting in enhanced presentation to Th cells as compared with nominal peptide (24, 25). In addition, recombinant Igs carrying B cell epitopes from the circumsporozoite or HIV Ags resulted in induction of neutralizing, anti-idiotypic responses (26, 27). Work using engineered recombinant Ig encompassing MHC class I-restricted epitopes showed that such epitopes may be processed by proteasome and presented via MHC class I by transfected cells to Tc (28). Overall, although these data support the concept that Id (or in general, Ig-associated Id) are being processed, presented, and recognized by immune cells, it is still unclear whether Ig bearing class I-restricted epitopes can elicit a Tc response. For example, transfectomas expressing recombinant Ig with a class I epitope from the nucleoprotein (NP) of influenza virus were capable of inducing, in certain conditions, a CTL response, whereas the purified Ig was unable to do so (28, 29). It has been thus concluded that the FcR-initiated processing pathway does not result in presentation of Id via MHC class I, due to a lack of intersection with the endogenous pathway of processing and presentation. More recent studies encompassing Ig carrying epitopes outside the idiotypic region (30) or immune complexes (IC), suggested otherwise and in the latter case, showed a therapeutic benefit in a preclinical tumor model as well as pinpointing the role of various classes of FcR in this process (31). Nevertheless, the practicality and potential safety profile of IC as therapeutic agents is uncertain.
In the present study, we systematically approach the question as to whether idiotypic class I-restricted determinants are immunogenic, using the model of a dominant NP epitope from the influenza virus and leveraging the potential of certain TLR ligands to promote APC activation. Further, we delineate the parameters regulating the magnitude and nature of the MHC class I-restricted response against the foreign epitope inserted within an Ig, in addition to the possibility of using such constructs to induce antitumoral responses capable of controlling malignant processes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
BALB/c, FcR–/– (32), and FcRIIB–/– mice (33) on a BALB/c background, and C.B10 congenic mice bearing the H-2b MHC haplotype on a BALB/c genetic background were purchased from The Jackson Laboratory and maintained at Alliance Pharmaceutical according to Institutional Animal Care and Use Committee and National Institutes of Health policies, in pathogen-specific free conditions. For all experiments, 8- to 12-wk-old female mice were used.
Reagents and cell lines
The dominant MHC class I (Kd)-restricted influenza virus NP epitope (147–155, TYQRTRALV) and the MHC class II (I-Ed)-restricted hemagglutinin (HA) epitope (110–120, SFERFEIFPKE) were previously described and well characterized (25, 34). Recombinant idiotypic Igs, IgNP and IgHA, bearing the NP and HA epitopes, respectively, were engineered by replacing the H chain CDR3 segments of a mouse IgG mAb with an original specificity against the hapten arsonate (23, 28). This manipulation retained binding to FcR, which is critical in effective targeting of APC (23, 25). The recombinant Ig peptides were obtained by cell culture and purified using affinity chromatography (23, 25, 28, 29).
The strain of influenza virus used in this study was A/WSN/32 H1N1, with mouse as permissive host. The virus was grown on Madine Darby Bovine Kidney carcinoma cells and purified, and titers were measured by conventional techniques (35). Synthetic dsRNA (pA:pU), previously characterized as a T1 adjuvant (36), was obtained from Sigma-Aldrich.
SP2/0 myelomatous cells, of MHC class I+ of H-2d background, widely used as partners for cell fusions in generating B cell hybridomas were previously described (37). Tumorigenic cells expressing secreted or retained Ags were obtained by transfection, selection, and subcloning of SP2/0 cells with plasmids carrying H chain and L chain of IgNP, IgHA, the IgG2b backbone, or whole-length influenza virus NP (encompassing a nuclear targeting motif) (38). Cells were characterized as previously described and the production of Ag confirmed by standard immunochemistry techniques. The mouse breast carcinoma cell line 4T-1 of BALB/c background were purchased from American Type Culture Collection, transfected with whole length NP, selected, subcloned, and characterized similarly. All cell lines were tested for tumorigenicity in BALB/c mice and upon retrieval were shown to retain Ag expression. Cells were cultured at 37°C under 5% CO2, FCS-free HL-1 medium (BioWhittaker) supplemented with HEPES and antibiotics.
Tumor challenge, monitoring, and immunotherapy
The mice were challenged by s.c. injection into the lateral back area, with 5 x 105 SP2/0 myeloma cells or 4T-1 breast carcinoma cells expressing TAA or TAA– control cells in 200 µl of DMEM. When the primary tumor became clinically detectable (day 12 or before if the tumor reached a volume of 0.5 ml) the mice were randomized into treatment groups. The treatment regimen was promptly initiated and repeated twice every 5 days, consisting of s.c. homolateral injection of 50 µg of pA:pU, 50 µg of Ig peptides, or a combination of 50 µg of pA:pU plus 50 µg of Ig peptides. The evolution of the primary tumor, closely paralleling the clinical progression, was monitored on a regular basis and the size of the tumor measured using a caliper (the volume of quasispherical tumors was estimated using the formula 4/3
((a+b)/2)3, in which a and b are two measured perpendicular diameters expressed in cubic centimeters. Mice displaying significant morbidity associated with terminal disease were euthanized.
Cell-based immunization was conducted using ex vivo Ag-loaded DC from animals having the same MHC haplotype. CD11c+ DC (2 x 106 cells/ml) purified by magnetic sorting according to manufacturer’s instructions (Miltenyi Biotec) were pulsed for 14 h with 50 µg/ml IgNP or an equivalent amount on a molar basis of NP peptide (1 µg/ml), in the presence or absence of 100 µg/ml dsRNA (pA:pU). After washing, these Ag-loaded DC were injected into recipient mice at 5 x 105 cells/mouse i.p. in 200 µl of DMEM.
Measurement of T cell response
For ELISPOT analysis, spleens were harvested and cell suspensions prepared using a standard technique involving removal of RBC by hypotonic lysis. Splenocytes were incubated at 5 x 105 cells/well (and serial, two-fold dilutions) in "complete" HL-1 medium (containing 10% FBS) with 10 µg/ml peptide (NP or HA) or medium only, in anti-IFN-, anti-IL-2, or anti-IL-4 (BD Pharmingen) precoated plates (MAHA S4150; Millipore), subsequently blocked with BSA. The cells were incubated for 72 h at 37°C and under 5% CO2. The assay was developed by first washing the cells, then serial incubation with 2 µg/ml biotin-conjugated anti-cytokine Abs (BD Pharmingen), followed by addition of streptavidin-conjugated HRP (1/1000 v/v) and AEC (3-amino-9-ethyl-carbazole) insoluble substrate (Sigma-Aldrich). The data corresponding to the number and average size of spot forming colonies were acquired using an automated system equipped with a camera (Navitar) and assisted by ImagePro Plus software (Media Cybernetics).
Cytokine production was measured after ex vivo stimulation of splenocytes, obtained as discussed, with 10 µg/ml peptide and in the presence of 5 U/ml rIL-2 for 5 days. The concentration of IFN- in the supernatants was measured by ELISA (BioSource International). The procedure was repeated following another round of stimulation with mitomycin-treated feeder cells, in presence of peptide and rIL-2 (same concentration as discussed), for 5 additional days.
For infection, mice were mildly anesthetized with isoflurane and treated by nasal instillation with a sublethal inoculum of 103 TCID50 (tissue culture infectious dose 50%) of WSN virus. The cytotoxic assay was carried by using effector cells from mice immunized with influenza virus Ags, obtained following ex vivo stimulation with 10 µg/ml NP and 5 U/ml rIL-2 for 5 days. The effector cells were coincubated, at various ratios, with peptide-coated or uncoated M12 (Kd+, B cell lymphoma) target cells for 5 h. Cell supernatants were tested for LDH release, an indicator of cellular damage, using a kit according to the manufacturer’s instructions (Cytotox 96, nonradioactive cytotoxicity assay kit; Promega).
CD8+ T cell separation, adoptive transfer, and FACS analysis
CD8+ T cells were isolated by magnetic selection from spleens harvested from mice that underwent tumor rejection subsequent to immunotherapy. The magnetic selection was conducted using beads coated with anti-CD8 mAbs according to the manufacturer’s instructions (Miltenyi) and the purity of CD8+ T cells (>95%) confirmed by flow cytometry (FACS) using a FACSCalibur instrument (BD Biosciences). Cells were resuspended at 10 x 106/100 µl and infused i.v. into syngeneic mice before challenge with tumor cells (100 µl/recipient).
For the characterization of the degree of activation of tumor-infiltrating lymphocytes, tumor masses were retrieved, collagenase digested, and cell suspensions prepared. The expression of CD25 was tested in CD3+ tumor-infiltrating lymphocytes, by using two-color FACS analysis, with reagents from BD Pharmingen.
Statistical analysis
Comparative analysis of the ELISPOT results was conducted by applying the t test, with values of p calculated accordingly. In addition, the log-rank test was used to analyze the tumor progression data.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
-producing T cells against an Ig-borne epitope
Effective antiviral and antitumoral immunity is thought to require generation of IFN- producing T cells and MHC class I-restricted cytotoxic cells. We studied the possibility of inducing MHC class I-restricted T cell responses by targeted Ag delivery via FcR expressed on professional APC. To circumvent the safety concerns posed by polyvalent Ag Ab IC, we used a molecule comprising the IgG2b backbone with a defined influenza virus-derived Kd-restricted epitope NP (147–155) (34) inserted within the CDR3 of the H chain IgNP (28).
We first assessed whether ex vivo epitope targeting of APC results in cross-processing and enables DC to trigger class I-restricted immunity. To this aim, CD11c+ DC isolated from secondary lymphoid organs were pulsed with IgNP or molar equivalent amount of NP peptide, washed, and adoptively transferred into naive BALB/c mice. As depicted in Fig. 1, C and E, IgNP or NP peptide-pulsed APC elicited IL-2- and IL-4-producing NP 147–155-reactive T cells, but no significant IFN--producing T cell immunity. We hypothesized that the activation state of the DC may have been limiting their capability in inducing IFN--producing Tc subsequent to processing of IgNP. To address this question, we took advantage of the previous observation that synthetic dsRNA activate professional APC, resulting in rapid induction of IL-12 and TNF-
(36). Copulsing of DC with IgNP and pA:pU promoted substantial generation of IFN--producing, NP 147–155-specific T cells upon adoptive transfer into naive BALB/c mice (Fig. 1A), with decreased induction of IL-4-producing T cells relative to DC pulsed with IgNP alone (Fig. 1E). In addition, FcR-mediated delivery of NP via recombinant Ig, in conjunction with DC activation by pA:pU, afforded an increased expansion of IFN--producing, specific T cells upon peptide restimulation, reflected into a larger size of spot forming colonies (Fig. 1B). As expected, pulsing of DC with pA:pU alone or transfer of DC incubated only with media, did not result in generation of NP 147–155-specific T cell responses.
|
component (Fig. 2A). Coadministration of IgNP and pA:pU greatly enhanced the generation of IFN-- and IL-2-producing NP 147–155-specific T cells with a limited impact; however, on IL-4-producing cells (Fig. 2A). In contrast, the administration of IgNP together with, or without pA:pU to CB10 congenic mice sharing the H-2b MHC haplotype with BALB/c, failed to induce NP 147–155 peptide specific immunity (data not shown).
|
-producing T cells, triggered by IgNP administration together with pA:pU, maintained their ability to produce IFN- upon repeated ex vivo stimulation with peptide and rIL-2, while in the presence of syngeneic APC (Fig. 2B). In contrast, the T cells induced by IgNP alone, failed to acquire the in vitro ability to produce IFN- when treated similarly. Nevertheless, NP 147–155-specific T cells primed in vivo with IgNP rapidly (within 4 days) acquired the capability to produce IFN- upon infection with influenza virus (Fig. 2C). The highest secondary expansion of IFN--producing T cells was measured shortly after infection of mice primed with a combination of IgNP and pA:pU (Fig. 2C). This was mirrored by more effective, early expansion of specific CTL in these mice (Fig. 2C) together with significantly decreased pulmonary virus titers (5-fold), showing that the generation of CTL effectors through cross-priming is regulated by the degree of APC activation.
Thus, dsRNA stimulation of APC was key to the induction of IFN--producing T cell immunity against an MHC class I-restricted epitope, incorporated within a recombinant Ig.
dsRNA-mediated cross presentation of recombinant Ig induces IFN--producing T cells effective against tumor growth
We next tested whether induction of MHC class I-restricted T cells by in vivo targeted delivery of a model tumor-associated epitope to FcR+ APC can control a tumor process in a preclinical model.
The tumor model used was based on the observation that s.c. inoculation of BALB/c mice with Kd+ SP2/0 myelomatous cells results in development of large primary tumor mass, progressing to metastasis to the major internal organs (liver, spleen), and significant mortality within 4 wk followed by death within 6–8 wk, concordant with the previously reported tumorigenic potential of hybridomas derived from SP2/0 cells (39). SP2/0 cells stably transfected with a plasmid expressing IgNP were previously shown to process and present the MHC class I-restricted NP 147–155 epitope in context of Kd (28, 29) and were used as model tumor cells. The s.c. inoculation of SP2/0-IgNP tumor cells into BALB/c mice resulted in progressive development of a primary tumor, metastasis, and death, similar to injection of nontransfected SP2/0 (Fig. 3A). We assessed whether immunotherapy of tumor-bearing mice with IgNP with or without pA:pU had any effect on evolution of tumor process. Mice displaying primary tumors of at least 0.5 cc were randomized into different treatment groups and received a series (5-day interval starting on day 12 or whenever tumor size reached 0.5 cc) of homolateral s.c. injections of 50 µg of pA:pU (Fig. 3B), 50 µg of IgNP (Fig. 3C), or 50 µg of IgNP plus 50 µg of pA:pU (Fig. 3D). Separate injection of IgNP or pA:pU resulted in a slight, nonstatistically significant slow-down in the growth of the primary tumor, with inexorable progression to serious morbidity followed by death. Tumor remission in naive, pA:pU or IgNP-treated mice was extremely rare (
10% or less). In contrast, the combination of IgNP and pA:pU resulted in significant control of primary tumor growth, induction of complete tumor regression, and prevention of serious morbidity and mortality in 60% of treated mice (Fig. 3D). The rest of the IgNP plus pA:pU treated mice displayed nonprogressing tumor disease with only 20% progressing to advanced disease resulting in moribund status. A similar trend was noted when the animals were treated with 10-fold lower doses of IgNP and pA:pU (5 µg), although most of the animals displayed stable primary tumor size rather than complete regression, consistent with a dose-effect relationship (data not shown).
|
R-mediated Ag loading along with activation of APC may be used to control tumor processes of diverse cell lineage origin.
Overall, these results suggest that the ineffective control of MHC class I epitope-expressing tumors may be due to reduced access of Ag to APC (immune ignorance) and/or to a limited degree of APC activation resulting in immune deviation or lack of differentiation to effector cells. To study this aspect, we measured the T cell reactivity to NP 147–155 peptide in spleens of nontreated mice that failed to control the tumor process and mice undergoing remission subsequent to immunotherapy with IgNP plus pA:pU. Interestingly, nontreated mice inoculated with SP2/0-IgNP cells developed a specific response confined to Tc2 cells (Fig. 4), similar to treated mice that failed to resolve the tumor. This development shows that TAA is transferred from tumor to APC, in vivo, in the absence of immunotherapy, illustrating the impact of cross-priming in this case. In contrast, the mice that underwent tumor rejection showed peptide-specific T cells comprising significant IFN-- and IL-2-producing subsets, along with IL-4 secreting cells (Fig. 4). Characterization by FACS analysis of tumor-infiltrating lymphocyte revealed a local infiltration with 
T cells (20–25%) and 
T cells (45–50%, with equal distribution between the CD4+ and CD8+ T cell subsets), irrespective of whether the mice were treated. In addition, the treated mice displayed higher frequency of IL-2R+ lymphocytes within tumors (on average, 5.9% in treated vs 0.8% in nontreated mice). In contrast to the mice treated with the combination of IgNP and pA:pU, mice treated with IgNP or pA:pU separately and unable to reject the tumor, presented an IL-4-biased profile, largely similar to nontreated mice (data not shown). Thus, in this model, immunotherapy with FcR-targeted epitope and APC activator (pA:pU) enabled the induction of Tc1 immunity and tumor rejection.
|
Induction of persisting immunity, with capability to counteract mechanisms of immune escape (such as Ag-loss) is of paramount importance to successful immunotherapy. Thus, we next tested whether the immune response subsequent to immunotherapy with IgNP plus pA:pU that elicited tumor rejection, conferred protection against subsequent challenge with homologous tumor. Mice that underwent remission were challenged with a tumorigenic dose of SP2/0-IgNP cells and followed for 1 mo. As shown in Fig. 5A, the challenged mice were completely protected against secondary challenge, whereas the control mice developed tumors with a 100% rate. Interestingly, mice that underwent immunotherapy and recovered from the primary SP2/0-IgNP tumor were completely protected against the challenge with SP2/0 myeloma cells expressing Ag variants (whole NP, IgHA encompassing an MHC class II-restricted influenza virus HA epitope, or IgG2b backbone) (Fig. 5, B, D, and E). In addition, such animals that recovered from primary tumor disease were protected against "loss-of-Ag" variant of the original tumor (untransfected SP2/0 myeloma cells), indicating that the repertoire of antitumoral T cells expanded during the process of tumor rejection gradually involving T cells specific for SP2/0 myeloma-associated epitopes in addition to the model TAA (NP). Ex vivo cultured T cells from animals resistant against multiple tumor variants display a long lasting, increased production of IFN-, IL-2, and IL-4 concordant with expanded frequency of antitumor effector cells, but hampering the efforts to define a specificity pattern (data not shown). Nevertheless, the mice resistant to SP2/0 myeloma antigenic variants, lacked protection against the Kd+ epithelial carcinoma cell line 4T-1 (Fig. 5F). Thus, there was no statistically significant difference between the evolution of 4T-1 carcinoma in naive vs mice recovered from SP2/0 tumor subsequent to immunotherapy. To evaluate the role of CD8+ T cells in this process of acquired broad immunity against SP2/0 Ags, adoptive transfer experiments were performed in mice challenged with SP2/0 or SP2/0-IgNP cells. In both cases, CD8+ T cells transferred from mice recovering from SP2/0-IgNP tumor upon immunotherapy with IgNP plus pA:pU, negatively interfered with the progression of tumoral process in recipient mice (Fig. 5G).
|
Contrasting roles of ITAM+ FcR and ITIM+ FcRIIB in controlling the antitumor T cell immunity elicited by recombinant Ig and dsRNA
Various FcR isoforms may be differentially involved in the response to IgNP plus pA:pU, FcR plus FcRI/RIII bear activating ITAM motifs and FcRIIB carries inhibitory ITIM motifs. For example, DC express FcRI and FcRIIB receptors and it is not clear how they participate following the response initiated by FcR targeted delivery of an MHC class I-restricted peptide. To address this issue, we induced tumors by inoculating SP2/0-IgNP myeloma cells into FcR–/– or FcRIIB–/– mice, along with FcR competent BALB/c mice. The mice underwent a similar treatment (5-day interval starting with day 12 or whenever tumor size reached 0.5 cc) with IgNP plus pA:pU, as in the previous experiments. At 4 wk after tumor initiation, disease evolution in treated FcRIIB–/– mice was similar to that of treated FcR-competent mice, with substantial and statistically significant control of tumor growth and prevention of morbidity, as opposed to nontreated control wild-type mice (Fig. 6A). In stark contrast, mice deficient in ITAM+ FcR that underwent treatment with IgNP plus pA:pU failed to control the tumor growth, similar to nontreated wild-type or FcR–/– mice (Fig. 6A). Even in the absence of treatment, there was a trend of FcRIIB–/– mice to control the tumoral growth, in contrast to wild-type or FcR–/– mice. That was complemented by increased frequency of IL-2+ NP-specific T cells in untreated FcRIIB–/– mice carrying SP2/0-IgNP tumors (Fig. 6C).
|
R genes upon immunotherapy with IgNP + pA:pU. In the absence of ITAM+ FcR, the immunotherapy failed to trigger substantial expansion of cytokine producing T cells (except, to a certain extent, IL-4+ T cells) along with inability to suppress the growth of the tumor (Fig. 6). In contrast, in the absence of functional ITIM+ FcRIIB, the treated mice mounted significantly stronger NP peptide-specific T cell responses along with effective control of the tumoral process. In fact, the expansion of cytokine-producing (in particular IFN-) T cells recognizing the MHC class I-restricted NP peptide was more substantial in treated FcRIIB–/– mice as compared with wild-type mice that underwent similar immunotherapy (Figs. 6 and 7). The ELISPOT analysis showed that in addition to the frequency, the secondary clonotypic expansion of NP 147–155-specific T cells were substantially and differentially influenced by FcRIIB and FcR+ receptors (Fig. 7).
|
R are similarly involved in the regulation of the immune response to MHC class II-restricted epitopes borne by CDR of IgG, we measured the immune response to APC loaded with IgHA using a protocol similar to that described for Fig. 1. As shown in Fig. 8, the FcR expression profile on APC influenced profoundly the magnitude of peptide-specific immune response subsequent to adoptive transfer of ex vivo IgHA-pulsed APC into naive BALB/c mice. In the absence of ITIM+ FcR expression, the resulting HA-specific response was significantly enhanced, mirroring the pattern observed in the case of IgNP. Thus, the FcRIIB–/– APC loaded with IgHA and cotreated with pA:pU triggered overall enhancement of both Th2 and IFN--producing Th1 cells (Fig. 8). In contrast, the magnitude of the immune response was diminished considerably in the absence of functional ITAM+ Fc receptors, or when the Ag was HA peptide alone rather than IgHA.
|
R subunit (essential component of the FcRI and FcRIII) is essential for the FcR targeted immunotherapy to be effective in this model of MHC class I-restricted TAA. In addition, these results show that ITAM+ and ITIM+ FcR regulate, in opposing fashion, the T cell response to class I- and class II-restricted epitope borne by CDR. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
Using models based on transplantable TAA+ tumors of lymphoid and carcinoma origin, it was shown that recombinant IgNP (carrying a class I-restricted NP determinant) (28) induced a strong immune response mirrored by a therapeutic effect (Fig. 3). However, this effect occurred only if IgNP was coadministered in conjunction with a potent activator of APC such as synthetic dsRNA (pA:pU) (38), resulting in a majority of mice recovering from the disease in the case of the SP2/0 myeloma tumor or with a stabilized disease in the case of the 4T-1 carcinoma (Fig. 3). No significant therapeutic effect was provided by either IgNP or dsRNA alone (Fig. 3); in addition, there was no effect of IgNP plus pA:pU on NP-negative SP2/0 tumor (data not shown). Measurement of immune response elicited by IgNP-pulsed DC in BALB/c mice showed that, although the recombinant Ig was more immunogenic than a molar equivalent amount of NP peptide (Fig. 1), the response was dominated by IL-4- and IL-2-producing T cells (for simplicity and with the caveat that the functional profile of MHC class I-restricted T cells is quite heterogenous, we will use the Tc1/Tc2 denomination to illustrate IFN-/IL-2- and IL-4-producing T cells, respectively). In contrast, coactivation of DC by pA:pU enabled induction of TcIFN- without interfering with the induction of TcIL-2, but resulting in slight diminution of the TcIL-4 population (Fig. 1). This profile was essentially reproduced by direct immunization with IgNP (Fig. 2): in contrast to IgNP alone, coadministration of pA:pU promoted induction of TcIFN- cells specific for the Id NP, with a stable cytokine profile (Fig. 2B), shifting the T1/T2 balance in favor of the former (Fig. 2A, inset). Interestingly, copriming with IgNP and pA:pU enabled a more rapid expansion of NP-specific TcIFN- and acquisition of cytolytic function shortly after infection with influenza virus (Fig. 2C). In addition, mice bearing SP2/0-IgNP tumors and undergoing tumor rejection subsequent to immunotherapy with IgNP plus pA:pU displayed increased numbers of NP-specific Tc-producing IFN- and IL-2, compared with tumor-bearing untreated mice (Fig. 4). In contrast, untreated mice (Fig. 4) or those failing to control the tumoral process subsequent to immunotherapy showed a predominance of TcIL-4 specific for the NP epitope. Together, these data show that coactivation of APC using a ligand for a TLR enables an optimal immunotherapeutic effect of the recombinant idiotypic Ig, resulting in differentiation of naive Tc to a stage encompassing TcIFN- with effector function relative to the tumoral process expressing secreted or retained Ag. Finally, in the absence of APC activation by TLR ligand, the MHC class I-restricted Id is still recognized, but the response is of limited magnitude and dominated by Tc2 (TcIL-4) and unable to mediate in vivo clearance of IgNP or NP-expressing cells.
In light of the fact that these tumor cells produce immunogenic Ig, one may have expected that pA:pU alone would have resulted in protective immunity against the tumor process. To address this question, mice challenged with tumor cells were injected with pA:pU alone. As shown in Fig. 3B, there was no significant beneficial effect on the tumor progression, conferred by pA:pU alone. Our interpretation was that the tumor process alone does not ensure optimal presentation of tumor-associated Ag in this system, despite the production of IgNP by tumor cells. This was further supported by the modest NP-specific response in nontreated, tumor-bearing mice similar to the modest Tc2 response in treated mice that failed to control the tumoral process (Fig. 4). Finally, based on the clear correlation between induction of T1 response and tumor regression (Fig. 4), we inferred that there are two limiting factors related to the antitumoral response in this system: first, the suboptimal Ag processing/presentation of endogenous tumor Ag and secondly, the status of innate immunity. Thus, to ensure a significant impact on tumor progression, an immunotherapeutic strategy must address both.
A crucial parameter of any immunotherapeutic strategy is to initiate pleiotropic effector mechanisms and/or induce immune cells recognizing an increased number of TAAs, counteracting immune escape mechanisms deployed by genetically unstable tumor cells. In addition, beyond effects on primary tumor, a successful immunotherapeutic strategy should mobilize immune effectors that have the capability to mediate a body-wide immune surveillance and curb metastatic disease. To address these questions, we tested whether mice that recovered from SP2/0 tumoral disease subsequent to immunotherapy can deal with further tumorigenic challenges comprising TAA variants (such as Ag-loss mutants). The striking, complete protection against subsequent ectopic challenge with antigenic variants of SP2/0 but not a different tumor cell line (Fig. 5, A–F), demonstrates that secondary immunity against SP2/0 TAA determinants occurred in an effective fashion, in the animals recovering from primary tumor due to treatment with IgNP plus pA:pU. Despite the fact that we cannot rule out at this point the involvement of additional immune effector mechanisms, adoptive transfer experiments strongly suggested a role for CD8+ T cells recognizing additional MHC class I-restricted, SP2/0-derived epitopes (Fig. 5G). Overall, these data show that during the immune effector process elicited by immunotherapy with the recombinant idiotypic Ig (Fig. 4D), a significant process of epitope spreading from the NP-determinant to additional epitopes borne by the tumor cells occurred, resulting in lasting protection against ectopic tumors comprising antigenic variants.
In addition to the level of APC activation determined by exposure to a TLR ligand, the response to the class I-restricted determinant borne by IgNP was significantly and differentially regulated by ITAM+ and ITIM+ Fc receptors. More specifically, intact functionality of ITAM+ FcR was critical for effective containment of the TAA+ tumoral process by immunotherapy with IgNP plus pA:pU (Fig. 6A). This result was paralleled by a decreased magnitude of immune response (both Tc1 and Tc2) against NP in tumor-bearing treated mice defective in the ITAM FcR subunit (Fig. 6, B–D). In contrast, although functionality of ITIM+ FcRIIB receptor was not required for effective tumor control by idiotypic immunotherapy (Fig. 6A), mice defective for FcRIIB showed an elevated Tc1 immunity against the MHC class I-restricted NP epitope (Figs. 6, B and C, and Fig. 7). Strikingly, a similar pattern was noticed in the case of an MHC class II-restricted epitope (HA 110–120 of influenza virus) borne by the CDR3 of the H chain of IgG (Fig. 8). Although ITAM+ Fc receptors were essential for the generation of Th immunity against HA, ITIM+ FcRIIB–– mice showed strongly enhanced Th1 and Th2 immunity upon immunization with IgHA. Together, these data show that beyond the receptor-mediated internalization resulting in more effective Ag processing and presentation in context of MHC class I or class II, ITAM+ and ITIM+ Fc receptors control the magnitude and quality of response against Id, likely by contributing to the regulation of APC maturation and function (40, 41). Further, ITIM+ Fc receptors constitute a checkpoint keeping under control the response against T cell Id. Additional studies are warranted, to outline elucidate whether selective targeting of ITAM+ FcR or inhibition of ITIM+ FcR circumvents the need to simultaneously activate APC in context of active immunotherapeutic approaches.
Together, these results have direct implications in regard to the immunotherapy of Id+ malignancies such as B cell lymphomas, myelomas, and some lymphocytic leukemias, using autologous idiotypic Ag. First, optimal cell based therapy with ex vivo pulsed DC must encompass activated APCs, and use of TLR ligands such as synthetic dsRNA or CpG motifs may accomplish this goal. This finding may explain why previous clinical studies with ex vivo pulsed immature DCs, despite achieving the goal of inducing moderate anti-Id responses, showed quite a limited clinical impact (17). Secondly, use of IgG as a vector is a far more optimal means to deliver epitopes to the APC for processing and presentation in the context of MHC class I or II molecules than is use of peptide epitopes, per se, for active efficient immunotherapy. This confirms prior observations with MHC class II epitopes (23, 25) and extends them to Tc epitopes; in both cases, FcRs bearing the subunit (ITAM+) mediate both cellular internalization and the amplification of subsequent APC function. Further, recombinant Ig may be safer and more practical than polyvalent IC for in vivo immunotherapy. Besides potential use of autologous Id for treatment of lymphatic malignancies, engineered recombinant Ig bearing epitopes (such as class I-restricted) derived from TAA may be used in combination with ligands for TLRs, such as synthetic dsRNA, for the treatment of solid tumors (e.g., carcinomas) expressing TAA. In addition, this type of agent may be used to build up or prime immunity against conserved epitopes associated with infectious agents of great concern for public health, such as influenza virus. Finally, interfering with the function of ITIM+ Fc receptors by various means (e.g., blocking Abs, peptidomimetics, antagonists interfering with downstream ITIM-dependent cell signaling) may greatly enable immunotherapy with Id+ autologous or recombinant Ig, or even enable immune systems to mount effective anti-Id responses as monotherapy. Conversely, selective interference with the function of ITAM+ Fc receptors in disorders mediated by IC (e.g., lupus nephritis) or amplification of the activity of ITIM+ Fc receptors (40) may represent a viable strategy alone or in conjunction with therapeutics addressing other pathogenic factors.
On a theoretical level, it becomes evident that although FcR-mediated internalization of Id+ Ig results in processing and presentation of MHC class I-restricted epitopes, the nature of immune response depends on the context of immunization (e.g., degree of APC activation, serving as a "checkpoint"). This explains apparent discrepancies with a previous report showing failure of induction of cytolytic immunity by IgNP (28) and cautions against exclusive use of a single readout for the measurement of MHC class I responses. Instead, this study and a previous study (42) unravel a great heterogeneity in regard to the phenotype and multiplicity of Tc subsets, with distinct function and potential, coexisting rather than being mutually exclusive and thus in accordance to recently proposed models (42, 43). In the absence of significant APC activation, idiotypic T cell determinants are being processed and presented but the immune response is limited and dominated by T2 cells. This study sheds light on the importance of a second checkpoint regulating this process, which is dependent on ITIM+ Fc receptors.
The existence of the idiotypic network was proposed more than three decades ago (6). Since then, advances in molecular mechanisms of generation of immune receptor diversity (4) have shed light on the nature of Id (4, 44). Besides the practical implications, this current work may unravel a mechanism explaining the homeostasis of the idiotypic network or management of the extraordinary idiotypic diversity of the Ig receptors. Thus, rather than being excluded from the repertoire via immune-mediated censorship, new Id (essential neo-self Ags) are actually immune monitored (Id processed and presented without significant response in a fashion resulting in clonal deletion, by virtue of the two checkpoints discussed previously). This finding implies that in a default mode, the idiotypic network has a reduced activity level dominated by regulatory/suppressive functions and, in contrast, appropriate APC activation may actually determine a transition to a high activity mode consisting in elevated anti-Id responses. This model (Fig. 9) is corroborated by previous evidence in humans showing immunization with Id resulting in a B cell, Th or Tc cell response (reviewed in Ref.17), implying that antigenic Id are not censored but rather managed in a way to minimize a negative impact on the diversity of the immune repertoire. Moreover, the Ig-VH region, and especially the CDR2-FR3-CDR3 area, encompasses multiple MHC class I- and II-restricted epitopes, frequently mirrored by low level T cell reactivity in multiple myeloma patients (45), making the occurrence of Id– Ig an extremely improbable event. It thus becomes evident that, due to the apparently high frequency of antigenic Id in the human immune repertoire, a censorship or negative selection of Id+ lymphocytes would heavily interfere with the diversity of the immunologic potential, suggesting that this homeostatic mechanism is of great importance to the functionality of the immune system.
|
In conclusion, the present study shows that recombinant Ig carrying disease-associated MHC class I-restricted epitopes are promising therapeutic tools. In addition, it delineates factors that must be dealt with to optimize their therapeutic use, enhancing in parallel our understanding on how idiotypic diversity is being managed via APC and the balance between ITAM+ and ITIM+ Fc receptors.
|
Disclosures |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Footnotes |
|---|
1 Address correspondence and reprint requests to Dr. Adrian Bot at the current address: Mannkind Corporation, 28903 North Avenue Paine, Valencia, CA 91355. E-mail address: abot{at}mannkindcorp.com 
2 Abbreviations used in this paper: TAA, tumor-associated Ag; CDR, complementarity-determining region; DC, dendritic cell; NP, nucleoprotein; HA, hemagglutinin; IC, immune complex.
Received for publication June 1, 2005. Accepted for publication November 8, 2005.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
) and second round (
) of peptide stimulation, was measured by ELISA (results expressed in picogram per milliliter as mean ± SEM of quadruplicate samples. C, To assess induction of CTL, mice were primed with IgNP alone or in combination with pA:pU and challenged 14 days later with infectious influenza virus, strain A/WSN/32 H1N1. Shortly after infection (4 days), the mice were sacrificed and the NP peptide-specific T cell response measured by ELISPOT analysis and cytotoxicity. The results were expressed as the number of IFN-
) vs the subgroup that recovered completely from tumoral disease, which represented 60% of the animals (
