Invariant Peptide Research Articles

Traumatic brain injury alters the effects of class II invariant peptide (CLIP) antagonism on chronic meningeal CLIP + B cells, neuropathology, and neurobehavioral impairment in 5xFAD mice

BackgroundTraumatic brain injury (TBI) is a significant risk factor for Alzheimer’s disease (AD), and accumulating evidence supports a role for adaptive immune B and T cells in both TBI and AD pathogenesis. We previously identified B cell and major histocompatibility complex class II (MHCII)-associated invariant chain peptide (CLIP)-positive B cell expansion after TBI. We also showed that antagonizing CLIP binding to the antigen presenting groove of MHCII after TBI acutely reduced CLIP + splenic B cells and was neuroprotective. The current study investigated the chronic effects of antagonizing CLIP in the 5xFAD Alzheimer’s mouse model, with and without TBI.Methods12-week-old male wild type (WT) and 5xFAD mice were administered either CLIP antagonist peptide (CAP) or vehicle, once at 30 min after either sham or a lateral fluid percussion injury (FPI). Analyses included flow cytometric analysis of immune cells in dural meninges and spleen, histopathological analysis of the brain, magnetic resonance diffusion tensor imaging, cerebrovascular analysis, and assessment of motor and neurobehavioral function over the ensuing 6 months.Results9-month-old 5xFAD mice had significantly more CLIP + B cells in the meninges compared to age-matched WT mice. A one-time treatment with CAP significantly reduced this population in 5xFAD mice. Importantly, CAP also improved some of the immune, histopathological, and neurobehavioral impairments in 5xFAD mice over the ensuing six months. Although FPI did not further elevate meningeal CLIP + B cells, it did negate the ability of CAP to reduce meningeal CLIP + B cells in the 5xFAD mice. FPI at 3 months of age exacerbated some aspects of AD pathology in 5xFAD mice, including further reducing hippocampal neurogenesis, increasing plaque deposition in CA3, altering microgliosis, and disrupting the cerebrovascular structure. CAP treatment after injury ameliorated some but not all of these FPI effects.

Abstract 58: Immunocytokines with specificity for invariant region of CAR molecules enhance effector function of multiple CAR T cell therapies

Abstract The effector functions of CAR T cells can be enhanced through delivery of immunostimulatory cytokines, such as IL-12, IL-18, or IL-23. However, systemic delivery of recombinant cytokines or paracrine secretion by CAR T cells can elicit toxicities due to non-specific targeting of the cytokines. Here, we designed immunocytokines with antibodies specific for the invariant linker peptides that join variable domains in the CAR scFv and immunostimulatory cytokines. The immunocytokines were capable of targeting CAR molecules agnostic of target antigen, demonstrating broad applicability without the need for further genetic engineering of CAR therapies, such as co-expression of a surrogate molecule (tEGFR, etc.). The addition of immunocytokines enhanced CAR T cell cytolytic activity and secretion of type I cytokines, even at low concentrations of immunocytokine. In a stringent Nalm6 xenograft model, the addition of immunocytokines delivering IL-12 enhanced the anti-tumor activity of anti-CD19 CAR T cells and overall survival compared to controls of antibody alone or PBS. This technological development presents the opportunity to specifically enhance the activity of multiple CAR T cell therapies in the absence of non-specific toxicities. Citation Format: J T. Keane, Oula K. Dagher, Fang Liu, Avery D. Posey. Immunocytokines with specificity for invariant region of CAR molecules enhance effector function of multiple CAR T cell therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 58.

Altered Affect and Increased CLIP+ B cells in the Meningeal Lymphatics of 5xFAD mice are mitigated by antagonizing MHCII‐associated invariant chain (CLIP)

AbstractBackgroundDepression often occurs prior to Alzheimer’s disease (AD) and both depression and AD have been linked to immune mechanisms. A key role for activated B cells in AD pathogenesis was recently identified. The study found that B cell depletion reduced Aβ plaque accumulation, neuroinflammation, and behavioral deficits in three AD transgenic mouse models. Our work has shown that treatment with a peptide antagonist for Major histocompatibility complex class II (MHCII) invariant peptide (CLIP), a key component in the transition between innate and adaptive immunity, results in selective depletion of pro‐inflammatory, CLIP+ve B cells and neuroprotection. We hypothesized that selective depletion of CLIP+ve B cells in the 5xFAD model of AD would reduce depression‐associated behavioral deficits.Method11‐week‐old male WT and 5xFAD mice underwent social interaction and burrowing pre‐testing. 12‐week‐old mice were treated once with a competitive antagonist peptide (CAP) or vehicle, followed by social interaction and burrowing testing once a month for 5 months. 6 months post‐treatment, meningeal lymphatics were collected, stained, and analyzed flow cytometrically for markers of B cells (CD19), T cells (CD3), and CLIP.ResultAffective alterations were observed in 5xFAD mice as early as 11 weeks of age and persisted up to 9 months of age. CAP treatment in 5xFAD mice protected against an age‐associated decline in social interaction. At 9 months of age 5xFAD mice were found to have significantly more B cells and CLIP+ve B cells in the meningeal lymphatics when compared to age‐matched WT, and CLIP+ve B cells were significantly decreased by a single CAP injection at 3 months of age.ConclusionA key function of B cells is their role as antigen presenting cells (APCs). CLIP occupies the MHCII antigen binding groove until it is displaced by antigen to be presented to CD4+ T cells, marking the transition between an innate and an adaptive immune response. Our findings suggest that 5xFAD mice exhibit depression‐associated behavior by 11 weeks of age, that CLIP+ve cells are elevated in 5xFAD mice, and that antagonizing CLIP depletes these B cells and functionally protects mice genetically predisposed to AD.

Intervention with VG1177, a MHC Class II Invariant Peptide Chain (CLIP) Antagonist, Restores Afferent Arteriolar Autoregulatory Behavior during Chronic 14 Day Lipopolysaccharide Treatment

Lipopolysaccharide (LPS) is a cell wall component of gram‐negative bacteria that is a known ligand for toll‐like receptor 4, which, in turn, activates the innate immune system. MHC Class II‐associated invariant peptide (CLIP) is critically linked to antigen processing, presentation, and adaptive immune system activation. Chronic immune system activation and inflammation are linked to blunted afferent arteriolar autoregulatory behavior and kidney injury. Previous work established that afferent arteriolar autoregulatory behavior is impaired following 7 days of LPS treatment by osmotic minipump, while simultaneous co‐treatment with VG1177 preserved autoregulatory capability. Accordingly, we postulated that competitive CLIP antagonist (VG177) treatment could rescue autoregulatory behavior impaired by chronic low‐dose LPS exposure. Osmotic minipumps were implanted (day 0) for infusion of LPS (0.01mg/kg/day) or saline (0.9% NaCl) for 14 days and then kidneys were harvested for juxtamedullary nephron studies. Three groups (n=6/group) were studied: saline, LPS, and LPS + VG1177. LPS + VG1177 group received LPS infusion from day 0 through day 14 and then daily VG1177 treatment (3mg/kg/day; i.p.) was added on LPS‐infusion days 7–14. Systolic blood pressures were similar in all three on day 14 (124 ± 1 mmHg, 124 ± 2 mmHg, and 125 ± 2 mmHg, respectively). Afferent arteriole autoregulatory behavior was assessed in these groups by increasing perfusion pressure from 65 to 170 mmHg in 15 mmHg increments. Starting diameters were similar across all three groups (15.1 ± 1.2 μm; 14.5 ± 1.1 μm and 14.7 ± 1.6 μm, respectively). When perfusion pressure was increased from 65 to 170 mmHg, arteriolar diameter in the saline group decreased by 25 ± 1% (P<0.05; n=6), exhibiting normal pressure‐induced autoregulatory vasoconstriction. Afferent arteriolar diameters in LPS treated kidneys decreased by just 4.5 ± 2% over the same pressure range, indicating significant (P<0.05 vs. saline) loss of autoregulatory behavior. In the LPS + VG1177 intervention group, afferent arteriolar diameter decreased by 15.2 ± 1% (P<0.05 vs. LPS) over the same pressure range, signifying partial restoration of afferent arteriolar autoregulatory behavior. Plasma endotoxin was measured on day 14 by Pierce LAL chromogenic endotoxin quantitation kit in the saline, LPS, and LPS + VG1177 groups. Plasma endotoxin concentration in the saline group averaged 5.5 ± 0.6 EU/mL compared to 67.7 ± 10.4 EU/mL; (P<0.05; n=6) in the 14 day LPS alone group. In 14 day LPS + 7 days of VG1177 intervention, plasma endotoxin concentration was significantly lower (29.8 ± 3.5 EU/mL; P<0.05; n=6) compared with LPS alone. Collectively, these data support the hypothesis that CLIP antagonism with VG1177 restores afferent arteriolar autoregulatory behavior during chronic 14 day low dose LPS treatment and may reduce the plasma endotoxin load. CLIP antagonism may open novel therapeutic targets for inflammatory kidney disease.Support or Funding InformationThis study was funded by a Pre‐doctoral Fellowship from the AHA Greater Southeast Affiliate: 14PRE20460061 and NIH: DK44628, HL098135.

Computer-Assisted Vaccine Design by Analysis of Zika Virus E Proteins Obtained either from Humans or from Aedes Mosquitos

Information entropy (H) and predicted B cell epitope score (Bepipred) were determined for the envelope E protein of Zika viruses (ZIKV) isolated from infected humans and Aedes mosquitos with the aim of identifying E protein regions that may be useful as immunological targets of anti-ZIKV vaccines. Total H of mosquito origin E proteins was 4.2380 greater than that of E proteins of human origin, suggestive of constraints on ZIKV mutation in the human host. Seven invariant peptides (H=0.0) of length 10 amino acids, or greater, were identified. These peptide sequences where H=0.0 were screened for predicted epitopes. The seven invariant peptides were comprised of 93 amino acid residues, 31 of which demonstrated predicted B-cell epitopic activity. The predicted epitopic residues were distributed predominantly to 5 of the 7 invariant peptides. It is proposed that these 5 invariant (H=0) peptides in the E proteins of both human and Aedes mosquito ZIKV represent domains with constrained mutational/evolutionary potential and that epitopes predicted to reside in such invariant domains thus may be stable immunological targets for development of an anti-ZIKV vaccine.

Discovery of a Direct Ras Inhibitor by Screening a Combinatorial Library of Cell-Permeable Bicyclic Peptides

Cyclic peptides have great potential as therapeutic agents and research tools. However, their applications against intracellular targets have been limited, because cyclic peptides are generally impermeable to the cell membrane. It was previously shown that fusion of cyclic peptides with a cyclic cell-penetrating peptide resulted in cell-permeable bicyclic peptides that are proteolytically stable and biologically active in cellular assays. In this work, we tested the generality of the bicyclic approach by synthesizing a combinatorial library of 5.7 × 106 bicyclic peptides featuring a degenerate sequence in the first ring and an invariant cell-penetrating peptide in the second ring. Screening of the library against oncoprotein K-Ras G12V followed by hit optimization produced a moderately potent and cell-permeable K-Ras inhibitor, which physically blocks the Ras-effector interactions in vitro, inhibits the signaling events downstream of Ras in cancer cells, and induces apoptosis of the cancer cells. Our approach should be generally applicable to developing cell-permeable bicyclic peptide inhibitors against other intracellular proteins.

Abstract P056: MHC CLass II-associated Invariant Peptide (CLIP) Antagonism During Chronic Lipopolysaccharide Treatment Preserves Afferent Arteriolar Autoregulatory Behavior

Lipopolysaccharide (LPS) is a cell wall component of gram-negative bacteria that can activate toll-like receptor 4, which in turn, activates the innate immune system. Chronic immune system activation is linked to blunted afferent arteriolar autoregulatory behavior and kidney injury. MHC Class II-associated invariant peptide (CLIP) provides a critical step in antigen processing, presentation, and adaptive immune system activation. Accordingly, we postulated that treatment with a competitive CLIP antagonist (CAP) during chronic low-dose LPS exposure would preserve afferent arteriolar autoregulatory behavior. Rats were implanted with osmotic minipumps (day 0) for infusion of LPS (0.01mg/kg/day) or saline (0.9% NaCl; 0.5μl/hr) for 8 days and then the kidneys were harvested for juxtamedullary nephron studies. Four groups (n=6/group) were studied: Control, LPS, LPS + CAP and Sham + CAP. Both LPS + CAP and Sham + CAP groups were treated with CAP (3mg/kg/day; i.p.) on days 1-7. Autoregulatory behavior was assessed in these groups by increasing perfusion pressure in 15 mmHg increments from 65 to 170 mmHg. Starting baseline diameters were similar across the control (15.2 ± 1.2 μm), LPS (14.1 ± 1.0 μm), LPS + CAP (15.1 ± 1.0), and Sham + CAP (15.7 ± 0.6) groups. When perfusion pressure was increased from 65 to 170 mmHg, control and sham + CAP afferent arteriolar diameter decreased significantly by 26 ± 4% and 25 ± 2% (P<0.05), respectively. In contrast, afferent diameters from LPS treated kidneys decreased by just 5 ± 2% over the same pressure range indicating impaired afferent arteriolar autoregulatory behavior. In LPS + CAP treated kidneys, afferent arteriolar diameter decreased by 17 ± 2% (P<0.05) over the same pressure range signifying preservation of autoregulatory behavior. These data support the hypothesis that CLIP antagonism during chronic low dose LPS treatment preserves afferent arteriolar autoregulatory behavior. Inhibiting CLIP may open novel therapeutic targets for inflammatory kidney disease.

Identification of Invariant Peptide Domains within Ebola Virus Glycoprotein GP1, 2

Six invariant oligopeptide sequences of length 12 amino acids, or greater, were identified in GP1,2 of ZEBOV Zaire and SEBOV Sudan Ebola viruses. Five of these invariant peptides had positive scores for predicted B-cell epitope scores. In contrast, the amino acid positions of the remaining invariant peptide all displayed negative predicted epitope scores. Invariant regions of Ebola GP1,2 oligopeptides may reflect structural, functional and immunological constraints on the virus, and thus, may be useful as immunological and pharmacological targets.

Traumatic brain injury causes selective, CD74-dependent peripheral lymphocyte activation that exacerbates neurodegeneration.

IntroductionTraumatic brain injury (TBI), a significant cause of death and disability, causes, as in any injury, an acute, innate immune response. A key component in the transition between innate and adaptive immunity is the processing and presentation of antigen by professional antigen presenting cells (APCs). Whether an adaptive immune response to brain injury is beneficial or detrimental is not known. Current efforts to understand the contribution of the immune system after TBI have focused on neuroinflammation and brain-infiltrating immune cells. Here, we characterize and target TBI-induced expansion of peripheral immune cells that may act as potential APCs. Because MHC Class II-associated invariant peptide (CLIP) is important for antigen processing and presentation, we engineered a competitive antagonist (CAP) for CLIP, and tested the hypothesis that peptide competition could reverse or prevent neurodegeneration after TBI.ResultsWe show that after fluid percussion injury (FPI), peripheral splenic lymphocytes, including CD4+ and CD8+ T cells, regulatory T cells (Tregs), and γδ T cells, are increased in number within 24 hours after FPI. These increases were reversed by CAP treatment and this antagonism of CLIP also reduced neuroinflammation and neurodegeneration after TBI. Using a mouse deficient for the precursor of CLIP, CD74, we observed decreased peripheral lymphocyte activation, decreased neurodegeneration, and a significantly smaller lesion size following TBI.ConclusionTaken together, the data support the hypothesis that neurodegeneration following TBI is dependent upon antigen processing and presentation that requires CD74.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-014-0143-5) contains supplementary material, which is available to authorized users.

Abstract 095: CD74 Deficient Mice are Resistant to Toll-Like Receptor-Induced Preeclampsia

Preeclampsia (PE) is a pregnancy-specific hypertensive disorder characterized by vascular endothelial dysfunction and excessive immunity and inflammation. Activation of the dsRNA receptor Toll-like receptor 3 (TLR3) or the ssRNA receptor TLR7 elicits a pregnancy-dependent PE-like syndrome in mice by inducing a pro-inflammatory immune response. CD74 (MHC Class II invariant chain) acts as a chaperone for MHC Class II surface expression on immune cells during antigen presentation and is cleaved into Class II-Associated Invariant Peptide (CLIP) following polyclonal activation of immune cell TLRs. The presence of CLIP in the groove of MHC Class II prevents T cell-dependent death leading to persistent immune cell activation. We hypothesized that genetic deletion of CD74 and subsequent depletion of CLIP on immune cells prevents TLR-induced immune responses and the development of PE in mice. Pregnant WT and CD74 KO mice were given i.p. injections of normal saline (P), poly I:C (TLR3 agonist; P-PIC), or R837 (TLR7 agonist; P-R837) on gestational days 13, 15, and 17 and euthanized on day 18. P-PIC and P-R837 WT mice had significantly increased splenic levels of pro-inflammatory CD3+/gd T cells and plasma levels of the gd T cell-derived cytokines IFNg, TNFa, and IL-17 compared to P WT mice whereas P-PIC and P-R837 CD74 KO mice had significantly increased anti-inflammatory CD3+/gd T cells and no significant increases in plasma IFNg, TNFa, and IL-17 levels. P-PIC and P-R837 CD74 KO mice did not develop the hypertension (gd17 SBP in mmHg: P WT=102±3, P CD74 KO=100±3, P-PIC WT=147±4*, P-PIC CD74 KO=95±3, P-R837 WT=133±2*, P-R837 CD74 KO=97±1; *p<0.05 vs. P WT), endothelial dysfunction, proteinuria, or placental necrosis seen in P-PIC and P-R837 WT mice. In conclusion, CD74 is crucial for the development of TLR-induced PE-like symptoms in mice and CD74/CLIP depletion may be a promising therapeutic target for women with PE.

Cathepsin S inhibition suppresses systemic lupus erythematosus and lupus nephritis because cathepsin S is essential for MHC class II-mediated CD4 T cell and B cell priming

ObjectivesMajor histocompatibility complex (MHC) class II-mediated priming of T and B lymphocytes is a central element of autoimmunity in systemic lupus erythematosus (SLE) and lupus nephritis. The cysteine protease cathepsin...

Regulation of HLA-DR peptide occupancy by histone deacetylase inhibitors

Numerous molecular effects have been attributed to histone deacetylase inhibitors (HDACI’s), including the induction of major histocompatibility (MHC) genes. Here we report that one FDA approved HDACI, Vorinostat, and a second HDACI currently in clinical trials, Entinostat, reduce the ratio of class II associated invariant peptide (CLIP) to the MHC class II molecule, HLA-DR, indicating an increase in the non-CLIP peptides bound to HLA-DR. The HDACI effects are apparent with immortalized B-cells, HLA-DR constitutive melanoma cells and with melanoma cells expressing HLA-DR due to transformation with an expression vector for the HLA-DR gene co-activator, CIITA. Entinostat treatment leads to upregulation of Cathepsin L1, and the HLA-DR peptidome of the Entinostat treated cells is consistent with increased Cathepsin L1 mediated proteolysis. These results indicate that HDACI treatments may alter the HLA-DR peptidome of cells in patients and provide a way to identify novel immunogens for vaccinations and the study of autoantigens.

Abstract 173: Depletion of CLIP+ Immune Cells Prevents Toll-Like Receptor-Induced Preeclampsia in Mice

Preeclampsia (PE) is a pregnancy-specific disorder of vascular endothelial dysfunction and inflammation which manifests as hypertension and proteinuria and may result from maladaptation of the maternal immune system. Activation of the dsRNA receptor Toll-like receptor 3 (TLR3) or the ssRNA receptor TLR7 elicits pregnancy-dependent hypertension and endothelial dysfunction in mice in part by increasing pro-inflammatory immune cells. Polyclonal activation of immune cells is associated with expression of Class II-Associated Invariant Peptide Chain (CLIP) in MHC class II which prevents T cell-dependent death leading to persistent immune cell activation. We hypothesized that removal of CLIP reduces pro-inflammatory immune cells and prevents the development of PE in mice. Pregnant mice were given ip injections of normal saline (P), poly I:C (TLR3 agonist; P-PIC), or R837 (TLR7 agonist; P-R837) with or without TPP, a custom-targeted peptide that removes CLIP from MHC class II, on gestational days 13, 15, and 17 and euthanized on day 18. TPP treatment significantly decreased pro-inflammatory CD3+/γδ T cells and increased anti-inflammatory CD4+/CD25+ regulatory T cells in TLR3-induced PE mice as well as TLR7-induced PE mice. TPP treatment also prevented the hypertension (GD17 SBP in mmHg: P=102±3, P+TPP=105±4, P-PIC=147±4*, P-PIC+TPP=103±2, P-R837=133±2*, P-R837+TPP=106±4; *p<0.05 vs. P), proteinuria, and endothelial dysfunction in TLR3-induced and TLR7-induced PE mice while having no detrimental effects in normal pregnant mice. Based on our results, the TPP-mediated decrease in detrimental immune cells positively modulated T cell subsets and prevented the development of PE-like symptoms in mice. CLIP removal from MHC class II with TPP may be a promising therapy for women with PE.

A Series of Human Cell-Based Artificial APC Expands Long-Lived, Th1-Biased, Viral Antigen-Specific CD4+ T Cells with a Central/Effector Memory Phenotpype Restricted by Common HLA-DR Alleles

AbstractAbstract 354Adoptive cell therapy utilizes unique mechanisms of action to prevent the development of infections in immunocompromised patients and treat chemotherapy resistant malignancies. In adoptive cell therapy, the major effector cells appear to be CD8+ T cells, since they are armed with antigen-specific effector functions, i.e. cytotoxicity and cytokine secretion. However, the roles of antigen-specific CD4+ T cells in T cell immunity are also critical. In immunocompromised patients adoptively transferred with CMV-specific CD8+ T cells, long-term in vivo persistence was achieved only when CMV-specific CD4+ T cells were also present in vivo. Recently, adoptive transfer of a NY-ESO-1 specific CD4+ T cell clone was reported to induce a complete response in a patient with metastatic melanoma. These results suggest that adoptive cell therapy for infectious diseases and cancer can be improved by infusing both antigen-specific CD4+ helper T cells as well as CD8+ CTL. Unfortunately, however, few versatile systems are available for producing large numbers of antigen-specific human CD4+ T cells for the purpose of adoptive therapy.K562 is a human erythroleukemic cell line, which lacks the expression of HLA class I and II, invariant chain (Ii), and HLA-DM, but does express adhesion molecules such as ICAM-1 and LFA-3. Given this unique immunologic phenotype, K562 has served as a useful tool in clinical cancer immunotherapy trials. Previously, we reported the generation of a K562-based artificial APC (aAPC), which expresses HLA-A2, CD80, and CD83. aAPC/A2 can uniquely support the priming and prolonged expansion of large numbers of antigen-specific CD8+ CTL which display a central/effector memory phenotype, possess potent effector function, and can be maintained in vitro without any feeder cells or cloning. aAPC/A2 is equipped with constitutive proteasome and inducible immunoproteasome machinery and can naturally process and present CD8+ T cell peptides via transduced A2 molecules. We have successfully generated clinical grade aAPC/A2 under cGMP conditions and conducted a clinical trial where patient with advanced melanoma are infused with large numbers of MART1-specific CTL generated ex vivo using aAPC/A2, IL-2 and IL-15.Based on our experience with aAPC/A2 and CD8+ T cells, we have generated a series of novel aAPC (aAPC/DR1, DR3, DR4, DR7, DR10, DR11, DR13, and DR15) to stimulate HLA-DR-restricted antigen-specific CD4+ T cells. K562 has been engineered to express HLA-DRα and β chains as a single HLA allele in conjunction with Ii, HLA-DMα and β chains, CD80 and CD83. CD83 delivers a CD80-dependent T cell stimulatory signal that allows T cells to be long-lived. Following the transduction of Ii, CLIP (class II invariant chain-associated peptide) appeared on the cell surface of aAPC. Furthermore, CLIP expression on aAPC was almost completely abrogated by the introduction of HLA-DM. This result is in accordance with previous studies showing that HLA-DM catalyzes the removal of CLIP from DR thus enabling exogenous peptides to bind to empty DR molecules in late endosomes. In addition to its endogenous pinocytic activity, aAPC was made capable of Fcγ receptor-mediated endocytosis by transduction of CD64. Comparison of naïve aAPC and CD64-transduced aAPC confirmed that Fcγ receptor-mediated endocytosis is more efficient than pinocytosis to take up soluble protein and process and present DR-restricted peptides to CD4+ T cells. Using these standardized and renewable aAPC, we determined novel viral protein-derived DR-restricted CD4+ T cell epitopes and expanded large numbers of viral antigen-specific CD4+ T cells without growing bystander Foxp3+ regulatory T cells. Without any feeder cells or cloning, expansion of CD4+ T cells using aAPC and low dose IL-2 and IL-15 was sustainable up to 150 days. Immunophenotypic analysis using HLA-DR tetramers and specific mAbs revealed that expanded CD4+ T cells were CD45RA−, CD45RO+, CD62L+-, demonstrating a central/effector memory phenotype. Furthermore, intracellular cytokine analysis showed that expanded DR-restricted viral-specific CD4+ T cells secreted IL-2 and IFN-γ but much less IL-4, displaying a Th1-biased phenotype. Taken all together, these results suggest that K562-based aAPC may serve as a translatable platform to generate both antigen-specific CD4+ helper T cells and CD8+ CTL. Disclosures:No relevant conflicts of interest to declare.

A label-free mass spectrometry method for the quantification of protein isotypes

Successful quantitative mass spectrometry (MS) requires strategies to link the mass spectrometer response to the analyte abundance, with the response being dependent on more factors than just analyte abundance. Label-dependent strategies rely on the incorporation of an isotopically labeled internal standard into the sample. Current label-free strategies (performed without internal standards) are useful for analyzing samples that are unsuitable for isotopic labeling but are less accurate. Here we describe a label-free technique applicable to analysis of products from related genes (isotypes). This approach enables the invariant tryptic peptide sequences within the family to serve as “built-in” internal standards and the isotype-specific peptide sequences to report the amount of the various isotypes. A process of elimination segregates reliably trypsin-released standard and reporter peptides from unreliably released peptides. The specific MS response factors for these reporter and standard peptides can be determined using synthetic peptides. Analysis of HeLa tubulin digests revealed peptides from βI-, βII-, βIII-, βIVb-, and βV-tubulin, eight of which were suitable; along with five standard peptides for quantification of the β-tubulin isotypes. To show the utility of this method, we determined that βI-tubulin represented 77% and βIII-tubulin represented 3.2% of the total HeLa β-tubulin.

Cysteine cathepsin S as an immunomodulatory target: present and future trends

Objective: Antigen presentation is the key to immune response. Cathepsin S plays a major role in the degradation of the invariant peptide chain associated with the major histocompatibility complex and thus affects antigen presentation. This review will focus on the recent developments made in field of cysteine cathepsins especially cathepsin S and their future prospects as a therapeutic target. Methods: Selective cathepsin inhibitors for targeting autoimmune disorders, atherosclerosis, osteoporosis, osteoarthritis and cancer are being pursued by many pharmaceutical companies. Recent publications in this field have been used as references to evaluate the current and future trends in cathepsin S inhibitors as an immunomodulatory target. Conclusions: The temporal and spatial position occupied by cathepsin S in immune presentation, gives rise to the hope that an inhibitor would impart selectivity with a lesser propensity for side effects than other immunosuppressive agents.

Clinical Outcomes of Patients with Recurrent/Refractory Hodgkin Disease Receiving Cyclosporine, Interferon-γ, and Interleukin-2 Immunotherapy To Induce Autoreactivity after Autologous Stem Cell Transplantation with BEAM: A COG Study.

The major risk for patients with recurrent or refractory Hodgkin Disease (HD) who receive standard treatment with high-dose chemotherapy and autologous hematopoietic stem cell rescue (ASCR) is relapse. However, in HD, the allogeneic graft-vs.-lymphoma effect is offset by high transplant-related mortality. The Children's Oncology Group has completed a pilot study of immunotherapy using cyclosporine, interferon-γ, and interleukin-2 to induce autologous graft-vs.-host disease (auto GVHD) in an effort to reduce the relapse rate without excessive morbidity and mortality. From 11/2003 to 2/2005, 24 patients with biopsy-confirmed recurrent/refractory HD were enrolled at 13 COG stem cell transplant institutions. One patient was inevaluable because of errors in protocol administration. At initial diagnosis, 5 were stage IV, 8 stage III, 7 stage II, 1 stage 1, and 3 unknown; 16 had B symptoms, and 15 had bulky disease. All patients had failed intensive multi-agent chemotherapy, and most had received prior radiation therapy. High risk patterns of relapse included extranodal disease (8), B symptoms (3), and bulky disease (2). We have previously presented the tolerability of this regimen, with the expected reversible complications and 4% non-relapse mortality. Moreover, we detected circulating autoreactive lymphocytes in vitro from the majority of patients during immunotherapy. These autoreactive lymphocytes respond to the HLA class II invariant peptide (CLIP). We therefore isolated these cells from selected patients by flow cytometry after staining with CLIP-DR2 tetramers, and demonstrated high perforin expression in CD8+ effector cells early in the course of immunotherapy, and a subsequent rise of FOXP3 expression in CD4+ regulatory T cells later in the course of immunotherapy. Importantly, CLIP was strongly expressed on the surface of Hodgkin Reed-Sternberg cells, as demonstrated by monoclonal antibody LN2 immunostaining of tumor samples from 8/9 patients for whom adequate tissue was submitted. To date, only 6 relapses have been reported. Updated event-free and overall survival data will be presented. We conclude that this immunotherapy regimen is tolerable, induces autoreactivity in a majority of patients, targets a molecule expressed consistently on Hodgkin Reed-Sternberg cells, and has produced encouraging clinical outcomes. We will proceed to test the efficacy of this immunotherapy regimen by randomizing patients with chemosensitive recurrent/refractory HD to receive immunotherapy or not after BEAM and ASCR.

Conformational Analysis of Invariant Peptide Sequences in Bacterial Genomes

The functional significance of evolutionarily conserved motifs/patterns of short regions in proteins is well documented. Although a large number of sequences are conserved, only a small fraction of these are invariant across several organisms. Here, we have examined the structural features of the functionally important peptide sequences, which have been found invariant across diverse bacterial genera. Ramachandran angles (phi,psi) have been used to analyze the conformation, folding patterns and geometrical location (buried/exposed) of these invariant peptides in different crystal structures harboring these sequences. The analysis indicates that the peptides preferred a single conformation in different protein structures, with the exception of only a few longer peptides that exhibited some conformational variability. In addition, it is noticed that the variability of conformation occurs mainly due to flipping of peptide units about the virtual C(alpha)...C(alpha) bond. However, for a given invariant peptide, the folding patterns are found to be similar in almost all the cases. Over and above, such peptides are found to be buried in the protein core. Thus, we can safely conclude that these invariant peptides are structurally important for the proteins, since they acquire unique structures across different proteins and can act as structural determinants (SD) of the proteins. The location of these SD peptides on the protein chain indicated that most of them are clustered towards the N-terminal and middle region of the protein with the C-terminal region exhibiting low preference. Another feature that emerges out of this study is that some of these SD peptides can also play the roles of "fold boundaries" or "hinge nucleus" in the protein structure. The study indicates that these SD peptides may act as chain-reversal signatures, guiding the proteins to adopt appropriate folds. In some cases the invariant signature peptides may also act as folding nuclei (FN) of the proteins.

Persistence of the Antibody Response to the VlsE Sixth Invariant Region (IR6) Peptide ofBorrelia burgdorferiafter Successful Antibiotic Treatment of Lyme Disease

It has been suggested that a <4-fold decline in the immunoglobulin G (IgG) antibody response to the VlsE sixth invariant region peptide of Borrelia burgdorferi within 6 months after antibiotic treatment may indicate spirochetal persistence in Lyme disease. We studied the response to this peptide in 77 patients with early or late disease, for whom archival samples were available at the time of antibiotic treatment and approximately 6 months or years later. Eight (33%) of the 24 patients with early manifestations and 18 (86%) of the 21 patients with late manifestations had a <4-fold decline in IgG anti-VlsE titers approximately 6 months after successful antibiotic treatment. Of 32 additional patients, 13 (50%) with early manifestations and 5 (83%) with late manifestations still had positive anti-VlsE titers 8-15 years after successful antibiotic treatment. We conclude that persistence of the anti-VlsE antibody response for months or years after antibiotic treatment cannot be equated with spirochetal persistence in Lyme disease.

A novel single chain I-A b molecule can stimulate and stain antigen-specific T cells

Multimers of soluble major histocompatibility complex class I and II molecules have proven to be useful reagents in quantifying and following specific T cell populations. This study describes the design, generation, and characterization of a novel, single chain I-A b molecule which utilizes a unique linker derived from the murine invariant chain. A fragment of the invariant chain, residues 58–85, binds to a region proximal to the class II peptide binding groove and stabilizes occupancy of the class II invariant chain-associated peptide. We have utilized this fragment, replacing CLIP with the Eα peptide sequence, to lock the attached peptide into the class II binding groove. The single chain I-A b molecule was recognized by a panel of conformation-sensitive, I-A b-specific, monoclonal antibodies. Membrane-bound and soluble forms of the single chain I-A b stimulated an antigen-specific T cell hybridoma, and tetramers made from soluble monomers stained these cells. The unique features of this molecule may be useful in the design of recombinant T cell receptor ligands containing peptides with low affinity for MHC.