Fn14 Deficiency Ameliorates Anti-dsDNA IgG-Induced Glomerular Damage in SCID Mice.

Many studies have demonstrated that anti-dsDNA IgG is closely associated with lupus nephritis. Recently, it was found that activation of the fibroblast growth factor-inducible 14 (Fn14) signaling pathway damages glomerular filtration barrier in MRL/lpr lupus-prone mice. However, MRL/lpr mice have high titers of serum autoantibodies other than anti-dsDNA IgG. The aim of this study was to further explore the effect of Fn14 deficiency on anti-dsDNA IgG-induced glomerular damage in severe combined immunodeficiency (SCID) mice that have no endogenous IgG. Fn14 deficiency was generated in SCID mice. The murine hybridoma cells producing control IgG or anti-dsDNA IgG were intraperitoneally injected into mice. In two weeks, the urine, serum, and kidney tissue samples were harvested from mice at sacrifice. It showed that the injection of anti-dsDNA IgG, but not control IgG hybridoma cells, induced proteinuria and glomerular damage in SCID mice. Between the wild-type (WT) and knockout (KO) mice injected with anti-dsDNA IgG hybridoma cells, the latter showed a decrease in both proteinuria and glomerular IgG deposition. The histopathological changes, inflammatory cell infiltration, and proinflammatory cytokine production were also attenuated in the kidneys of the Fn14-KO mice upon anti-dsDNA IgG injection. Therefore, Fn14 deficiency effectively protects SCID mice from anti-dsDNA IgG-induced glomerular damage.

Microencapsulated Cells for Cancer Therapy.

The microencapsulation of different types of cells that are able to produce therapeutic factors is being investigated for the treatment of several human diseases. Most efforts are focused on chronic and degenerative diseases as this strategy could become an alternative to some commonly used parenteral treatments that need to be repeatedly administered. But, this approach has also been investigated in the field of oncology with the aim of providing immunomodulatory antibodies that are able to enhance the patient's inherent immune response against the tumor. These kind of treatments would provide the patient with the therapeutic drug produced in situ, de novo, and in a sustained way, making the therapy more comfortable.Although different devices are nowadays available to produce cell-enclosing alginate-microcapsules, here, we describe the most important steps and advices in order to fabricate alginate-poly-L-lysine-alginate microcapsules containing hybridoma cells for cancer management using an electrostatic bead generator, and how to evaluate the viability of those cells over the time.

Antiviral CD8⁺ T cells cause an experimental autoimmune encephalomyelitis-like disease in naive mice.

Major histocompatibility complex class I-restricted CD8(+) cytotoxic T lymphocytes are involved in the pathogenesis of multiple sclerosis (MS) and both autoimmune, experimental autoimmune encephalomyelitis, and viral, Theiler's murine encephalomyelitis virus (TMEV) infection, animal models of MS. Following TMEV infection, certain T cell hybridomas, generated from cloned TMEV-induced CD8(+) T cells, were able to produce clinical signs of disease (flaccid hind limb paralysis) upon adoptive transfer into naive mice. Dual T cell receptors (TCR) are present on the surface of these cells as both Vß3 and Vß6 were detected by polymerase chain reaction (PCR) screening and flow cytometry and multiple Vα mRNAs were detected by PCR screening. This is the first demonstration of antiviral CD8(+) T cells having more than one TCR initiating an autoimmune disease in the natural host of the virus. We hypothesize that this is a potential mechanism for virus-induced autoimmune disease initiated by CD8(+) T cells.

Olfactory sensory axon growth and branching is influenced by sonic hedgehog.

Olfactory sensory neuron (OSN) axons extend from the olfactory epithelium to the olfactory bulb without branching until they reach their target region, the glomerulus. In this report, we present evidence to support the involvement of sonic hedgehog in promoting rat olfactory sensory axons to branch and to enter into the glomerulus. Sonic hedgehog (Shh) protein is detected in the glomeruli of the olfactory bulb, whereas its transcript is expressed in the mitral and tufted cells, suggesting that Shh in the glomeruli is produced by mitral and tufted cells. In primary OSN cultures, Shh-N peptide promotes olfactory axon branching. When Shh function is neutralized in vivo by its antibody, growth of newly generated OSN axons into the glomeruli is vastly reduced.

Preliminary report on tumor stem cell/B cell hybridoma vaccine for recurrent glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM), the most aggressive glioma, presents with a rapid evolution and relapse within the first year, which is attributed to the persistence of tumor stem cells (TSC) and the escape of immune surveillance. Mixed leukocyte culture (MLC) cytoimplant has been shown to function as a powerful intratumor pro-inflammatory cytokine pump. Tumor B-cell hybridoma (TBH) vaccines have been shown to function as antigen-presenting cells. We evaluated the toxicity and efficiency of each treatment alone and in combination. PATIENTS AND METHODS: In an open study, 12 consecutive patients were evenly divided into 3 groups, each group receiving 3 different treatments. Patients in Group 1 were treated, after diagnosis, with debulking surgery (DS)+radiotherapy (Rx), and after the first relapse underwent DS+MLC treatment. Patients in Group 2 were similarly treated but after the first relapse underwent DS+MLC+TBH. Finally, patients in Group 3 were similarly treated but after the first relapse underwent DS+TBH. Nestin PAP stain assessed TSC participation in TBH. RESULTS: Treatment with MLC had strong and rapid therapeutic effects, but was limited in duration and induced various degrees of brain inflammation. Treatment with MLC+TBH acted synergistically, provoking a rapid, strong and lasting therapeutic response but also generating different degrees of brain inflammation. A lasting therapeutic effect without generating high degrees of brain inflammation occurred in patients treated with TBH vaccine alone. CONCLUSION: TSC vaccine consisting of TBH alone seems to have potent adjuvant reactions overcoming both persistence of tumor stem cells and immune escape of GBM without provoking an encephalitic reaction.

Macrophage MHC receptor 2: a novel receptor on allograft (H-2D(d)K(d))-induced macrophage (H-2D(b)K(b)) recognizing an MHC class I molecule, H-2K(d), in mice.

We previously reported that a population of allograft (H-2D(d)K(d))-induced macrophages (AIM) in C57BL/6 (H-2D(b)K(b)) mice exhibited major histocompatibility complex (MHC) haplotype (H-2D(d)) specific killing of the allograft (e.g., BALB/c skin and Meth A cells; H-2D(d)K(d)) in a macrophage MHC receptor (MMR)-dependent manner. In the present study, we isolated a cDNA clone encoding a novel receptor (MMR2) on AIM recognizing another MHC class I molecule, H-2K(d), by the expression cloning method using H-2K(d) tetramer and a monoclonal antibody (mAb; R12) specific for AIM. The cDNA (2359-bp) encoded a 677-amino acid polypeptide of a calculated molecular mass of 87 kDa and was found to be expressed exclusively on AIM among cells infiltrating into allografts on days 0-9 after transplantation. Confocal microscopy showed that HEK293T cells transfected with this cDNA were reactive toward the H-2K(d) molecule but not toward other MHC class I molecules such as H-2D(d), H-2D(b), H-2D(k), H-2K(b), H-2K(k), and H-2L(d) molecules. The binding of the H-2K(d) molecule to the transfectants was inhibited by the addition of R12 or anti-H-2K(d), but not by R15 (a mAb specific for H-2D(d) receptor) or anti-H-2D(d), mAb. Flow cytometric analysis revealed specific binding of H-2K(d) molecules to AIM (K(d)=2.7x10(-9) M); and the binding was completely suppressed by the addition of R12 mAb. These results demonstrate that a novel receptor (MMR2) for H-2K(d) molecules was induced on effector macrophages responsible for allograft (H-2D(d)K(d)) rejection by H-2D(b)K(b) mice.

Developing the concept of adoptive cellular gene therapy of rheumatoid arthritis.

Progressive destruction of articular cartilage and bone is the pivotal problem of rheumatoid arthritis (RA). Joint destruction is the cause of severe disability and determines the long-term outcome of disease. Conventional therapy does not control this destructive process sufficiently and the anti-rheumatic drugs available today can cause severe systemic adverse effects. Local application of chondroprotective and osteoprotective agents by means of gene therapy would be an attractive alternative to conventional therapy of RA and could provide long-term expression of the therapeutic agents and minimize systemic adverse effects. For this purpose, we have developed the concept of adoptive cellular gene therapy. This treatment strategy is based on using genetically engineered cells that home specifically to sites of autoimmune inflammation and thus allow local delivery of therapeutic gene products. Ex vivo transduction of these cells avoids systemic exposure of the host to the transgene-encoding vector and thus adds to the safety of this approach. In this article of the CIS Spring School in Autoimmune Diseases 2005 proceedings, we review our work on developing the strategy of adoptive cellular gene therapy and summarize recent advances in the evaluation of therapeutic effects and the identification of novel therapeutic targets.

The role of complement in cryoglobulin-induced immune complex glomerulonephritis.

Many forms of glomerulonephritis are triggered by Ab localization in the glomerulus, but the mechanisms by which this induces glomerular inflammation are not fully understood. In this study we investigated the role of complement in a mouse model of cryoglobulin-induced immune complex glomerulonephritis. Several complement-deficient mice on a C57BL/6 and BALB/c genetic background were used and compared with strain-matched, wild-type controls. Cryoglobulinemia was induced by i.p. injection of 6-19 hybridoma cells producing an IgG3 cryoglobulin with rheumatoid factor activity against IgG2a of allotype a present in BALB/c, but not C57BL/6, mice. Thus, the cryoprecipitate in C57BL/6 mice consisted of the IgG3 cryoglobulin only (type I cryoglobulinemia) compared with IgG3-IgG2a complexes in BALB/c (type II cryoglobulinemia). The survival of mice was not affected by complement deficiency. Glomerular influx of neutrophils was significantly less in C3-, factor B-, and C5-deficient mice compared with wild-type and C1q-deficient mice. It did not correlate with C3 deposition, but did correlate with the amount of C6 deposited. Deficiency of CD59a, the membrane inhibitor of the membrane attack complex, did not induce an increase in neutrophil infiltration, suggesting that the generation of C5a accounts for the effects observed. There was no apparent difference between cryoglobulinemia types I and II regarding the role of complement. Our results suggest that in this model of cryoglobulin-induced glomerulonephritis the neutrophil influx was mediated by C5 activation with the alternative pathway playing a prominent role in its cleavage. Thus, blocking C5 is a potential therapeutic strategy for preventing renal injury in cryoglobulinemia.

Conditioning of neonatal pigs using low-dose chemotherapy and murine fetal tissue before murine hybridoma transplantation.

Immunosuppression, myeloablation, and the use of immunologically immature tissue can overcome major histocompatibility complex barriers by inducing tolerance. With the goal of inducing tolerance to BALB/c-derived murine hybridoma cells producing the 4C6 monoclonal antibody (mAb), we transplanted BALB/c fetal tissue into neonatal pigs during a regimen of low-dose conditioning with busulfan and cyclophosphamide. After the tolerance induction phase, animals received intraperitoneal injections of 4C6 mAb hybridoma cells. Evidence of persistence of injected cells over time was sought by molecular analysis of peripheral blood for the presence of mouse genomic sequences and circulating 4C6 mAb. Persistence of donor polymerase chain reaction signal during the entire duration of the study, detectable mAb titer for 6 weeks, and a twofold increase of mAb concentration after a boost hybridoma infusion was observed in one animal receiving six consecutive administrations of the conditioning regimen. Our model has the distinctive advantage of allowing functional monitoring of engrafted cells for studying tolerance induction strategies. In addition, this model could be the basis for approaches aimed at producing mAbs in tolerized large animals.

Back-pack mice as a model of renal mesangial IgA dimers deposition.

Mesangial IgA in IgA nephropathy are dimers with a J chain but no poly-Ig receptor. This molecular structure has led to the hypothesis that these IgA are issued from the lamina propria of mucosal areas, reaching the kidney by way of the peripheral blood. The availability of hybridomas producing IgA dimers provided an opportunity to test this hypothesis in a new experimental model of IgA nephropathy. Mice were injected subcutaneously (back-pack mice) or intraperitoneally with hybridoma cells secreting either monoclonal IgA dimers, or monoclonal IgA monomers. The influence of immune complex formation was also tested in both these models. Renal IgA deposition was investigated 12 days after the injection of hybridoma cells. Backpack mice developed highly vascularized subcutaneous tumors. Mesangial IgA deposits were observed only in dimeric IgA hybridoma back-pack animals. No significant staining was observed in glomeruli from animals injected with hybridoma cells producing monomeric IgA. None of the hybridomas induced mesangial deposition when injected intraperitoneally. This animal model demonstrates the capacity of circulating IgA dimers to spontaneously form mesangial deposits and contributes to confirm the involvement of abnormalities of mucosal immunity in the pathogenesis of IgA nephropathy.

Regenerating corticospinal fibers in the Marmoset (Callitrix jacchus) after spinal cord lesion and treatment with the anti-Nogo-A antibody IN-1.

Neutralizing the myelin-associated growth inhibitor Nogo-A in adult spinal cord-injured rats can promote regeneration of injured and compensatory sprouting of uninjured axons. Nogo-A is present in humans, making its neutralization a possible novel treatment option for paraplegic patients. In this study we examined the effects of an extensively used anti-Nogo-A antibody (mAb IN-1) on the regenerative capabilities of lesioned corticospinal tract (CST) axons in a primate, the Marmoset monkey. Unilateral thoracic lesions of the CST were performed in six adult Marmosets, followed by the application of mAb IN-1 into the cerebrospinal fluid circulation by a graft of hybridoma cells. A unilateral injection of biotin dextran amine into the motor cortex was performed to analyse sprouting and regeneration of the lesioned axons. In the control antibody-treated animal CST fibers stopped rostral to the lesion site and often showed retraction bulbs. In contrast, in four out of five mAb IN-1-treated animals fine labeled neurites had grown into, through and around the lesion site. Thus, this study provides first anatomical evidence that in primates, the neutralization of the myelin-associated inhibitor Nogo-A results in increased regenerative sprouting and growth of lesioned spinal cord axons.

Evidence that IgE molecules mediate a spectrum of effects on mast cell survival and activation via aggregation of the FcepsilonRI.

We demonstrate that binding of different IgE molecules (IgEs) to their receptor, FcepsilonRI, induces a spectrum of activation events in the absence of a specific antigen and provide evidence that such activation reflects aggregation of FcepsilonRI. Highly cytokinergic IgEs can efficiently induce production of cytokines and render mast cells resistant to apoptosis in an autocrine fashion, whereas poorly cytokinergic IgEs induce these effects inefficiently. Highly cytokinergic IgEs seem to induce more extensive FcepsilonRI aggregation than do poorly cytokinergic IgEs, which leads to stronger mast cell activation and survival effects. These effects of both types of IgEs require Syk tyrosine kinase and can be inhibited by FcepsilonRI disaggregation with monovalent hapten. In hybridoma-transplanted mice, mucosal mast cell numbers correlate with serum IgE levels. Therefore, survival effects of IgE could contribute to the pathogenesis of allergic disease.

Functional reorganization of the motor cortex in adult rats after cortical lesion and treatment with monoclonal antibody IN-1.

We previously reported anatomical plasticity in the adult motor cortex after a unilateral sensorimotor cortex (SMC) lesion and treatment with monoclonal antibody (mAb) IN-1, which permits neurite outgrowth from the intact, opposite cortex into deafferented subcortical targets. This study was designed to investigate whether treatment with the mAb IN-1 after SMC lesion in the adult leads to functional reorganization of the intact, opposite motor cortex. Adult rats underwent unilateral SMC aspiration lesion and treatment with either mAb IN-1 or control antibody, or no treatment. After a 6 week survival period, the intact, opposite forelimb motor cortex was explored using intracortical microstimulation to evoke forelimb movements. A dramatic increase in ipsilateral movements of the lesion-impaired forelimb was found in animals treated with mAb IN-1 compared with control animals. These results resembled our previous findings of cortical reorganization in the spared hemisphere after neonatal cortical lesion and without any additional treatment. These results show that, after adult cortical lesion, treatment with mAb IN-1 induces a functional reorganization of the intact, opposite motor cortex.

Antibody-mediated CNS demyelination II. Focal spinal cord lesions induced by implantation of an IgM antisulfatide-secreting hybridoma.

We showed previously that spinal cord implants of hybridoma cells (O1) that secrete an IgM antigalactocerebroside cause focal multiple-sclerosis-like plaques of demyelination followed by remyelination to form "shadow plaques" (Rosenbluth et al., 1999). The antibody in that case was directed against a glycolipid present in mature oligodendrocytes and myelin but not in precursor cells. We now report the effects of implanting a different hybridoma (O4) that secretes IgM antibodies directed against sulfatide, a constituent not only of mature myelin and oligodendrocytes but also of late precursor cells, in order to determine whether this hybridoma too would generate focal demyelination and would, in addition, block remyelination. Our results show that focal plaques of demyelination indeed appear after O4 implantation, and that remyelination does occur, but only in cases where the hybridoma cells have degenerated, probably through host rejection. The occurrence of remyelination suggests that oligodendrocyte precursor cells are capable of migrating in rapidly from adjacent areas or that early precursors, not yet expressing sulfatide, remain undamaged within the lesions. In cases where intact hybridoma cells persist at lesion sites, remyelination does not occur. Failure of remyelination in this model thus appears to result from the continuing presence of antimyelin antibodies rather than from depletion of oligodendrocyte precursors.

A transgenic mouse model of autoimmune glomerulonephritis and necrotizing arteritis associated with cryoglobulinemia.

Mice implanted with hybridoma secreting 6-19 IgG3 anti-IgG2a rheumatoid factor (RF) with cryoglobulin activity develop acute glomerulonephritis and cutaneous leukocytoclastic vasculitis. As the RF activity is implicated in the skin, but not glomerular lesions, it is still unclear whether the renal pathogenicity is determined by 6-19 H chains alone or their combination with L chains. To address this question, we have generated transgenic mice expressing only the H chain gene or both H and L chain genes of the 6-19 IgG3 anti-IgG2a RF and determined the development of glomerular and vascular lesions. H-single and H/L-double transgenic mice displayed comparable high amounts of IgG3 cryoglobulins, but only H/L-double transgenic mice having 10-fold higher levels of IgG3 anti-IgG2a RF progressively developed chronic, lethal glomerulonephritis. The severe glomerular lesions observed at 8-10 mo of age were very heterogeneous (membranoproliferative changes, crescents, and sclerosis); in addition, one-third of them had necrotizing arteritis in the kidneys and skeletal muscles. These renal and vascular changes were very different from those observed in the acute cryoglobulinemia, characterized by mainly "wire-loop" glomerular lesions and a cutaneous leukocytoclastic form of vasculitis. Thus, our data demonstrate the importance of a unique combination of the H and L chains for the expression of the pathogenic activity of IgG3 cryoglobulins and that a single autoantibody is able to induce different types of glomerular and vascular complications, depending on its production levels and kinetics.

Progesterone inhibits rejection of xenogeneic transplants in the sheep uterus.

OBJECTIVES: One of the proposed roles of progesterone is to prevent maternal immunological destruction of the allogeneic conceptus. Here, it was demonstrated that progesterone allows survival of a xenotransplant placed in the uterine lumen. METHODS: Ovariectomized ewes, surgically prepared to have ligatures around each uterine horn, were given daily subcutaneous injections of 50 mg progesterone or vehicle (sesame oil). After 30 days of treatment, mouse hybridoma cells were transplanted to one ligated uterine horn and phosphate-buffered saline was injected into the other horn. The uterus was flushed after an additional 14 days of treatment and hybridoma cells were identified by immunofluorescence. RESULTS: Overall, hybridoma cells were recovered from 4 of 5 progesterone-treated ewes and 1 of 5 vehicle-treated ewes. Immunohistochemical analysis of intercaruncular endometrium using antibodies towards CD8, gammadelta, and CD45R lymphocyte markers revealed that local presence of hybridoma cells caused a significant increase in CD8+ cells in all tissue compartments. While not significant, the numbers of CD8+ cells in the luminal and glandular epithelium were lower for progesterone-treated ewes. Progesterone tended to increase gammadelta T cell numbers in the glandular epithelium. CONCLUSIONS: Results demonstrate that xenograft rejection in the uterus is associated with an increase in CD8+ cells in the endometrium and that progesterone can inhibit uterine tissue graft rejection responses sufficiently to allow survival or delay rejection of xenograft tissue.

Engineered fusion hybrid vaccine of IL-4 gene-modified myeloma and relative mature dendritic cells enhances antitumor immunity.

Dendritic cell (DC)-tumor fusion hybrid vaccine which facilitates antigen presentation represents a new powerful strategy in cancer therapy. In the present study, we investigated the antitumor immunity derived from vaccination of fusion hybrids between wild-type J558 or engineered J558-IL-4 myeloma cells secreting cytokine interleukin-4 (IL-4) and immature DCs (DC(IMAT)) or relative mature DCs (DC(RMAT)). DC(RMAT) displayed an up-regulated expression of immune molecules (Ia(d), CD40, CD54, CD80 and CD86) and certain cytokines/chemokines, and enhanced ability of allogeneic T cell stimulation when compared to DC(IMAT). These DCs were fused with myeloma cells by polyethylene glycol (PEG). The fusion efficiency was approximately 20%. Our data showed that immunization of C57BL/6 mice with DC(RMAT)/J558 hybrids induced protective immunity against a high dose of J558 tumor challenge (1x10(6) cells) in 3 out of 10 immunized mice, compared with no protection seen in mice immunized with DC(IMAT)/J558 hybrids. Furthermore, immunization of mice with engineered DC(RMAT)/J558-IL-4 hybrids elicited stronger J558 tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro and induced more efficient protective immunity (10/10 mice; tumor free) against J558 tumor challenge in vivo than DC(RMAT)/J558 hybrid vaccines. The results demonstrate the importance of DC maturation in DC-tumor hybrid vaccines and indicate that the engineered fusion hybrid vaccines which combine gene-modified tumor and DC vaccines may be an attractive strategy for cancer immunotherapy.

Suppression of experimental lupus nephritis by aberrant expression of the soluble E-selectin gene.

Circulating leukocytes, particularly neutrophils and monocytes, are important effector cells in the induction of many forms of glomerulonephritis. Adhesion molecules, especially selectins, are also thought to be critical for the development of this disease. We examined the possible suppressive effect of soluble E-selectin on the development of experimental lupus nephritis induced by the injection of a hybridoma clone (2B11.3) derived from an MRL/MpJ-lpr/lpr lupus mouse. This clone produces IgG3 antibodies that induce severe proliferative glomerulonephritis resembling lupus nephritis when injected into normal mice. Transgenic mice with a soluble E-selectin gene were injected intraperitoneally with the hybridoma cells and histopathologically examined on day 15. As a result, the development of glomerulonephritis was significantly suppressed. This suppression was characterized by fewer inflammatory cell infiltrates, compared with non-transgenic litter mates, despite the fact that there were no remarkable differences in immunoglobulin deposits or expression of E-selectin between the two groups. These findings suggest that by controlling inflammatory cell infiltration, soluble E-selectin plays a preventative role in the development of a particular type of lupus nephritis.

Impact of the lymphoma idiotype on in vivo tumor protection in a vaccination model based on targeting antigens to antigen-presenting cells.

Trioma cell vaccination is a potent new immunologic approach for the therapy of malignant B-cell lymphoma. It is based on targeting tumor antigens to internalizing receptors on antigen-presenting cells (APCs). Tumor cells are fused to an APC-specific hybridoma, where they are converted to trioma cells that include potentially all lymphoma-derived antigens and that express the APC-binding arm. In this study, the mechanisms of trioma-mediated tumor immunity in immunocompetent mice were dissected, and it was shown in this model system that humoral anti-idiotypic immunity is indeed detectable after idiotype-specific immunization but that it does not reflect the degree of tumor protection obtained in vivo. Immunization against the idiotype alone was not sufficient for efficient tumor rejection in vivo. Targeting tumor antigens to APCs is only successful in terms of inducing tumor protection when designed as a polyvalent vaccination protocol.

Idiotype and anti-idiotype specific T cell responses on transplantation with hybridomas reactive to viral hemagglutinin and human tumor antigen.

B cell hybridomas expressing class I and II MHC molecules and producing antibodies directed against hemagglutinin protein of Rinderpest virus and human Mucin-1 have been used as surrogate B cells to study T cell responses against the antigens. The observed CTL and lymphoproliferative response indicates that anti-idiotypic B cells termed Jerne cells stimulate both T helper and T cytotoxic cells by virtue of their ability to present recycled or regurgitated peptido-mimics of antigen to T helper cells through class II MHC and de novo synthesized peptido-mimics of antigens to CTLs. Thus, T cell memory response can be perpetuated by anti-idiotypic Jerne B cells and these findings lend support to the earlier proposed relay hypothesis for perpetuation of immunological memory (IM).