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Neoantigen immunogenicity prediction is a highly challenging problem in the development of personalised medicines. Low reactivity rates in called neoantigens result in a difficult prediction scenario with limited training datasets. Here we describe ImmugenX, a modular protein language modelling approach to immunogenicity prediction for CD8+ reactive epitopes. ImmugenX comprises of a pMHC encoding module trained on three pMHC prediction tasks, an optional TCR encoding module and a set of context specific immunogenicity prediction head modules. Compared with state-of-the-art models for each task, ImmugenX's encoding module performs comparably or better on pMHC binding affinity, eluted ligand prediction and stability tasks. ImmugenX outperforms all compared models on pMHC immunogenicity prediction (Area under the receiver operating characteristic curve = 0.619, average precision: 0.514), with a 7% increase in average precision compared to the next best model. ImmugenX shows further improved performance on immunogenicity prediction with the integration of TCR context information. ImmugenX performance is further analysed for interpretability, which locates areas of weakness found across existing immunogenicity models and highlight possible biases in public datasets., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: S.A.Q. is co-founder and chief scientific officer and own shares in Achilles Therapeutics. C.S. acknowledges grants from AstraZeneca, Boehringer-Ingelheim, Bristol Myers Squibb, Pfizer, Roche-Ventana, Invitae (previously Archer Dx Inc., a collaboration in minimal residual disease sequencing technologies), Ono Pharmaceutical and Personalis. He is chief investigator for the AZ MeRmaiD 1 and 2 clinical trials and is the steering committee chair. He is also co-chief investigator of the NHS Galleri trial funded by GRAIL and a paid member of GRAIL’s scientific advisory board (SAB). He receives consultant fees from Achilles Therapeutics (also an SAB member); Bicycle Therapeutics (also an SAB member); Genentech; Medicxi; the China Innovation Centre of Roche (CICoR), formerly Roche Innovation Centre Shanghai; Metabomed (until July 2022); Relay Therapeutics; and the Sarah Cannon Research Institute. C.S has received honoraria from Amgen, AstraZeneca, Bristol Myers Squibb, GlaxoSmithKline, Illumina, MSD, Novartis, Pfizer and Roche-Ventana; previously held stock options in Apogen Biotechnologies and GRAIL; currently has stock options in Epic Bioscience and Bicycle Therapeutics; and has stock options and is co-founder of Achilles Therapeutics. S.R.H. is the cofounder of PokeAcell and is co-inventor of licensed patents related to T cell detection. H.O’B., M.S., L.M. and F.O’F. are employees of Achilles Therapeutics. H.O’B. and M.S. are both named inventors on patents for ImmugenX., (Copyright: © 2024 O’Brien et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

ADAPTeR is a prospective, phase II study of nivolumab (anti-PD-1) in 15 treatment-naive patients (115 multiregion tumor samples) with metastatic clear cell renal cell carcinoma (ccRCC) aiming to understand the mechanism underpinning therapeutic response. Genomic analyses show no correlation between tumor molecular features and response, whereas ccRCC-specific human endogenous retrovirus expression indirectly correlates with clinical response. T cell receptor (TCR) analysis reveals a significantly higher number of expanded TCR clones pre-treatment in responders suggesting pre-existing immunity. Maintenance of highly similar clusters of TCRs post-treatment predict response, suggesting ongoing antigen engagement and survival of families of T cells likely recognizing the same antigens. In responders, nivolumab-bound CD8 + T cells are expanded and express GZMK/B. Our data suggest nivolumab drives both maintenance and replacement of previously expanded T cell clones, but only maintenance correlates with response. We hypothesize that maintenance and boosting of a pre-existing response is a key element of anti-PD-1 mode of action., Competing Interests: Declaration of interests L.A. is funded by the Royal Marsden Cancer Charity. E.H. and M.M. are funded by Cancer Research UK (CRUK). F.B. is funded by the Rosetrees Trust (M829). J.A. is a full-time employee of Hoffmann-La Roche AG (Basel, Switzerland). D.A.M has received consultancy fees from AstraZeneca, Thermo Fisher, and Eli Lilly. A.F. has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 892360. L.P. has received research funding from Pierre Fabre, and honoraria from Pfizer, Ipsen, Bristol-Myers Squibb, and EUSA Pharma. R.S. has received non-financial support from Merck and Bristol Myers Squibb; research support from Merck, Puma Biotechnology, and Roche; and advisory board fees for Bristol Myers Squibb; and personal fees from Roche for an advisory board related to a trial-research project; all related to breast cancer research projects. R.S. reports no conflict of interests related to this project. M.J.H. is a Cancer Research UK (CRUK) Clinician Scientist (RCCFEL\100099) and has received funding from CRUK, National Institute for Health Research, Rosetrees Trust, UKI NETs and NIHR University College London Hospitals Biomedical Research Center. M.J.H. is a member of the Scientific Advisory Board and Steering Committee for Achilles Therapeutics. G.K. is a scientific co-founder of and consulting for Enara Bio and a member of its scientific advisory board. G.K. receives core funding from the Francis Crick Institute (FC0010099). B.C. is supported by a CRUK Project Grant. J.L. has received research funding from Bristol-Myers Squibb, Merck, Novartis, Pfizer, Achilles Therapeutics, Roche, Nektar Therapeutics, Covance, Immunocore, Pharmacyclics, and Aveo, and served as a consultant to Achilles, AstraZeneca, Boston Biomedical, Bristol-Myers Squibb, Eisai, EUSA Pharma, GlaxoSmithKline, Ipsen, Imugene, Incyte, iOnctura, Kymab, Merck Serono, Nektar, Novartis, Pierre Fabre, Pfizer, Roche Genentech, Secarna, and Vitaccess. C.S. acknowledges grant support from Pfizer, AstraZeneca, Bristol-Myers Squibb, Roche-Ventana, Boehringer-Ingelheim, Archer Dx Inc (collaboration in minimal residual disease sequencing technologies), and Ono Pharmaceutical, is an AstraZeneca Advisory Board member and Chief Investigator for the MeRmaiD1 clinical trial, has consulted for Pfizer, Novartis, GlaxoSmithKline, MSD, Bristol-Myers Squibb, Celgene, AstraZeneca, Illumina, Genentech, Roche-Ventana, GRAIL, Medicxi, Bicycle Therapeutics, and the Sarah Cannon Research Institute, has stock options in Apogen Biotechnologies, Epic Bioscience, GRAIL, and has stock options and is co-founder of Achilles Therapeutics. Patents: C.S. holds European patents relating to assay technology to detect tumor recurrence (PCT/GB2017/053289); to targeting neoantigens (PCT/EP2016/059401), identifying patent response to immune checkpoint blockade (PCT/EP2016/071471), determining HLA LOH (PCT/GB2018/052004), predicting survival rates of patients with cancer (PCT/GB2020/050221), identifying patients who respond to cancer treatment (PCT/GB2018/051912), a US patent relating to detecting tumor mutations (PCT/US2017/28013) and both a European and US patent related to identifying insertion/deletion mutation targets (PCT/GB2018/051892). C.S. is Royal Society Napier Research Professor (RP150154). His work is supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001169), the UK Medical Research Council (FC001169), and the Wellcome Trust (FC001169). C.S. is funded by Cancer Research UK (TRACERx, PEACE and CRUK Cancer Immunotherapy Catalyst Network), Cancer Research UK Lung Cancer Center of Excellence, the Rosetrees Trust, Butterfield and Stoneygate Trusts, NovoNordisk Foundation (ID16584), Royal Society Research Professorship Enhancement Award (RP/EA/180007), the NIHR BRC at University College London Hospitals, the CRUK-UCL Center, Experimental Cancer Medicine Center and the Breast Cancer Research Foundation, USA (BCRF). His research is supported by a Stand Up To Cancer-LUNGevity-American Lung Association Lung Cancer Interception Dream Team Translational Research Grant (SU2C-AACR-DT23-17). Stand Up To Cancer is a program of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the Scientific Partner of SU2C. C.S. also receives funding from the European Research Council (ERC) under the European Union’s Seventh Framework Program (FP7/2007-2013) Consolidator Grant (FP7-THESEUS-617844), European Commission ITN (FP7-PloidyNet 607722), an ERC Advanced Grant (PROTEUS) from the European Research Council under the European Union’s Horizon 2020 research and innovation program (835297), and Chromavision from the European Union’s Horizon 2020 research and innovation program (665233). S.A.Q. is a CRUK Senior Cancer Research Fellowship (C36463/A22246) and is funded by a CRUK Biotherapeutic Program Grant (C36463/A20764) and the Rosetrees and Stonygate Trusts (A1388) and a donation from the Khoo Teck Puat UK Foundation via the UCL Cancer Institute Research Trust (539288). S.T. is funded by Cancer Research UK (grant reference number C50947/A18176), the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC0010988), the UK Medical Research Council (FC0010988), and the Wellcome Trust (FC0010988), the National Institute for Health Research (NIHR) Biomedical Research Center at the Royal Marsden Hospital and Institute of Cancer Research (grant reference number A109), the Royal Marsden Cancer Charity, The Rosetrees Trust (grant reference number A2204), Ventana Medical Systems Inc (grant reference numbers 10467 and 10530), the National Institutes of Health (Bethesda, MD) and Melanoma Research Alliance. ST has received speaking fees from Roche, Astra Zeneca, Novartis, and Ipsen. S.T. has the following patents filed: Indel mutations as a therapeutic target and predictive biomarker PCTGB2018/051892 and PCTGB2018/051893 and Clear Cell Renal Cell Carcinoma Biomarkers P113326GB., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent clinical experience has demonstrated that adoptive regulatory T (Treg) cell therapy is a safe and feasible strategy to suppress immunopathology via induction of host tolerance to allo- and autoantigens. However, clinical trials continue to be compromised due to an inability to manufacture a sufficient Treg cell dose. Multipotent adult progenitor cells (MAPC Ⓡ ) promote Treg cell differentiation in vitro , suggesting they may be repurposed to enhance ex vivo expansion of Tregs for adoptive cellular therapy. Here, we use a Good Manufacturing Practice (GMP) compatible Treg expansion platform to demonstrate that MAPC cell-co-cultured Tregs (MulTreg) exhibit a log-fold increase in yield across two independent cohorts, reducing time to target dose by an average of 30%. Enhanced expansion is coupled to a distinct Treg cell-intrinsic transcriptional program characterized by elevated expression of replication-related genes ( CDK1, PLK1, CDC20 ), downregulation of progenitor and lymph node-homing molecules ( LEF1 CCR7, SELL ) and induction of intestinal and inflammatory tissue migratory markers ( ITGA4, CXCR1 ) consistent with expression of a gut homing (CCR7lo β 7 hi) phenotype. Importantly, we find that MulTreg are more readily expanded from patients with autoimmune disease compared to matched Treg lines, suggesting clinical utility in gut and/or T helper type1 (Th1)-driven pathology associated with autoimmunity or transplantation. Relative to expanded Tregs, MulTreg retain equivalent and robust purity, FoxP3 Treg-Specific Demethylated Region (TSDR) demethylation, nominal effector cytokine production and potent suppression of Th1-driven antigen specific and polyclonal responses in vitro and xeno Graft vs Host Disease (xGvHD) in vivo . These data support the use of MAPC cell co-culture in adoptive Treg therapy platforms as a means to rescue expansion failure and reduce the time required to manufacture a stable, potently suppressive product., Competing Interests: AT and AV-T are employees of Athersys Inc. and JB and VR are employees of ReGenesys BV a subsidiary of Athersys Inc. AT, AV-T, JB, and VR have compensated stock options from Athersys, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Reading, Roobrouck, Hull, Becker, Beyens, Valentin-Torres, Boardman, Lamperti, Stubblefield, Lombardi, Deans, Ting and Tree.)

Programmed cell death protein 1 (PD-1) is expressed on T cells upon T cell receptor (TCR) stimulation. PD-1 ligand 1 (PD-L1) is expressed in most tumor environments, and its binding to PD-1 on T cells drives them to apoptosis or into a regulatory phenotype. The fact that PD-L1 itself is also expressed on T cells upon activation has been largely neglected. Here, we demonstrate that PD-L1 ligation on human CD25-depleted CD4+ T cells, combined with CD3/TCR stimulation, induces their conversion into highly suppressive T cells. Furthermore, this effect was most prominent in memory (CD45RA-CD45RO+) T cells. PD-L1 engagement on T cells resulted in reduced ERK phosphorylation and decreased AKT/mTOR/S6 signaling. Importantly, T cells from rheumatoid arthritis patients exhibited high basal levels of phosphorylated ERK and following PD-L1 cross-linking both ERK signaling and the AKT/mTOR/S6 pathway failed to be down modulated, making them refractory to the acquisition of a regulatory phenotype. Altogether, our results suggest that PD-L1 signaling on memory T cells could play an important role in resolving inflammatory responses; maintaining a tolerogenic environment and its failure could contribute to ongoing autoimmunity., Competing Interests: The authors have declared that no competing interests exist.

B cells have been implicated in transplant rejection via antibody-mediated mechanisms and more recently by presenting donor antigens to T cells. We have shown in patients with chronic antibody-mediated rejection that B cells control the indirect T cell alloresponses. To understand more about the role of B cells as antigen-presenting cells for CD4 + T cell with indirect allospecificity, B cells were depleted in C57BL/6 mice, using an anti-CD20 antibody, prior to receiving MHC class I-mismatched (K d ) skin. The absence of B cells at the time of transplantation prolonged skin graft survival. To study the mechanisms behind this observation, T cells with indirect allospecificity were transferred in mice receiving a K d skin transplant. T cell proliferation was markedly inhibited in the absence of recipient B cells, suggesting that B cells contribute to indirect pathway sensitization. Furthermore, we have shown that a possible way in which B cells present alloantigens is via acquisition of MHC-peptide complexes. Finally, we demonstrate that the addition of B cell depletion to the transfer of regulatory T cells (Tregs) with indirect alloresponse further prolonged skin graft survival. This study supports an important role for B cells in indirect T cell priming and further emphasizes the advantage of combination therapies in prolonging transplant survival., (© 2020 The Authors. American Journal of Transplantation published by Wiley Periodicals LLC on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons.)

Tumour mutational burden (TMB) predicts immunotherapy outcome in non-small cell lung cancer (NSCLC), consistent with immune recognition of tumour neoantigens. However, persistent antigen exposure is detrimental for T cell function. How TMB affects CD4 and CD8 T cell differentiation in untreated tumours, and whether this affects patient outcomes is unknown. Here we paired high-dimensional flow cytometry, exome, single-cell and bulk RNA sequencing from patients with resected, untreated NSCLC to examine these relationships. TMB was associated with compartment-wide T cell differentiation skewing, characterized by loss of TCF7-expressing progenitor-like CD4 T cells, and an increased abundance of dysfunctional CD8 and CD4 T cell subsets, with significant phenotypic and transcriptional similarity to neoantigen-reactive CD8 T cells. A gene signature of redistribution from progenitor-like to dysfunctional states associated with poor survival in lung and other cancer cohorts. Single-cell characterization of these populations informs potential strategies for therapeutic manipulation in NSCLC., Competing Interests: Competing interest statement S.A.Q., K.S.P. and C.S. are co-founders of Achilles Therapeutics. C.S. receives grant support from Pfizer, AstraZeneca, BMS, Roche-Ventana and Boehringer-Ingelheim. C.S. has consulted for Pfizer, Novartis, GlaxoSmithKline, MSD, BMS, Celgene, AstraZeneca, Illumina, Genentech, Roche-Ventana, GRAIL, Medicxi, and the Sarah Cannon Research Institute. C.S. is a shareholder of Apogen Biotechnologies, Epic Bioscience, GRAIL, and has stock options in and is co-founder of Achilles Therapeutics. R.R., N.M. and G.A.W. have stock options in and have consulted for Achilles Therapeutics. J.L.R and M.A.B have consulted for Achilles Therapeutics. P.D.B and M.W.S are employees of Achilles Therapeutics.

CD8 + T cells provide a critical defence from pathogens at mucosal epithelia including the female reproductive tract (FRT). Mucosal immunisation is considered essential to initiate this response, however this is difficult to reconcile with evidence that antigen delivered to skin can recruit protective CD8 + T cells to mucosal tissues. Here we dissect the underlying mechanism. We show that adenovirus serotype 5 (Ad5) bio-distributes at very low level to non-lymphoid tissues after skin immunisation. This drives the expansion and activation of CD3 - NK1.1 + group 1 innate lymphoid cells (ILC1) within the FRT, essential for recruitment of CD8 + T-cell effectors. Interferon gamma produced by activated ILC1 is critical to licence CD11b + Ly6C + monocyte production of CXCL9, a chemokine required to recruit skin primed CXCR3 + CD8 + T-cells to the FRT. Our findings reveal a novel role for ILC1 to recruit effector CD8 + T-cells to prevent virus spread and establish immune surveillance at barrier tissues.

Repair of tissue damaged during inflammatory processes is key to the return of local homeostasis and restoration of epithelial integrity. Here we describe CD161 + regulatory T (T reg ) cells as a distinct, highly suppressive population of T reg cells that mediate wound healing. These T reg cells were enriched in intestinal lamina propria, particularly in Crohn's disease. CD161 + T reg cells had an all-trans retinoic acid (ATRA)-regulated gene signature, and CD161 expression on T reg cells was induced by ATRA, which directly regulated the CD161 gene. CD161 was co-stimulatory, and ligation with the T cell antigen receptor induced cytokines that accelerated the wound healing of intestinal epithelial cells. We identified a transcription-factor network, including BACH2, RORγt, FOSL2, AP-1 and RUNX1, that controlled expression of the wound-healing program, and found a CD161 + T reg cell signature in Crohn's disease mucosa associated with reduced inflammation. These findings identify CD161 + T reg cells as a population involved in controlling the balance between inflammation and epithelial barrier healing in the gut.

The superoxide-generating enzyme Nox2 contributes to hypertension and cardiovascular remodeling triggered by activation of the renin-angiotensin system. Multiple Nox2-expressing cells are implicated in angiotensin II-induced (Ang II-induced) pathophysiology, but the importance of Nox2 in leukocyte subsets is poorly understood. Here, we investigated the role of Nox2 in T cells, particularly Tregs. Mice globally deficient in Nox2 displayed increased numbers of Tregs in the heart at baseline, whereas Ang II-induced effector T cell (Teff) infiltration was inhibited. To investigate the role of Treg Nox2, we generated a mouse line with CD4-targeted Nox2 deficiency (Nox2fl/flCD4Cre+). These animals showed inhibition of Ang II-induced hypertension and cardiac remodeling related to increased tissue-resident Tregs and reduction in infiltrating Teffs, including Th17 cells. The protection in Nox2fl/flCD4Cre+ mice was reversed by anti-CD25 antibody depletion of Tregs. Mechanistically, Nox2-/y Tregs showed higher in vitro suppression of Teff proliferation than WT Tregs, increased nuclear levels of FoxP3 and NF-κB, and enhanced transcription of CD25, CD39, and CD73. Adoptive transfer of Tregs confirmed that Nox2-deficient cells had greater inhibitory effects on Ang II-induced heart remodeling than WT cells. These results identify a previously unrecognized role of Nox2 in modulating suppression of Tregs, which acts to enhance hypertension and cardiac remodeling.

The generation of tissue resident memory (T RM ) cells at the body surfaces to provide a front line defence against invading pathogens represents an important goal in vaccine development for a wide variety of pathogens. It has been widely assumed that local vaccine delivery to the mucosae is necessary to achieve that aim. Here we characterise a novel micro-needle array (MA) delivery system fabricated to deliver a live recombinant human adenovirus type 5 vaccine vector (AdHu5) encoding HIV-1 gag. We demonstrate rapid dissolution kinetics of the microneedles in skin. Moreover, a consequence of MA vaccine cargo release was the generation of long-lived antigen-specific CD8 + T cells that accumulate in mucosal tissues, including the female genital and respiratory tract. The memory CD8 + T cell population maintained in the peripheral mucosal tissues was attributable to a MA delivered AdHu5 vaccine instructing CD8 + T cell expression of CXCR3 + , CD103 +, CD49a + , CD69 + , CD127 + homing, retention and survival markers. Furthermore, memory CD8 + T cells generated by MA immunization significantly expanded upon locally administered antigenic challenge and showed a predominant poly-functional profile producing high levels of IFNγ and Granzyme B. These data demonstrate that skin vaccine delivery using microneedle technology induces mobilization of long lived, poly-functional CD8 + T cells to peripheral tissues, phenotypically displaying hallmarks of residency and yields new insights into how to design and deliver effective vaccine candidates with properties to exert local immunosurveillance at the mucosal surfaces., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Due to the clinical relevance of suicidal risks, suicide attempts, and suicides in adolescence consensus-based guidelines with clinical recommendations were updated and summarized in this article. It should be considered that each indication has to be taken serious. Suicidality is an essential component of the psychopathological report and should be explored for the short- and long term risk. The clinical assessment of the acute suicidality results from a trustful anamnestic conversation, assessment of risk factors, mental disorders, and the use of alcohol and drugs. Acute suicidality is an indication for an inpatient treatment that for the protection of the patient has to be implemented also against his will. An adequate documentation is inevitable. After a suicide attempt, in addition to the initial medical treatment it has to be paid attention that the patient cannot harm himself any further and a rapid consultation in the responsible clinic takes place. First therapeutic goal is the reduction of suicidality and if necessary the re-achievement of the ability to negotiate a non-suicide agreement. For recurrent suicide thoughts an emergency plan has to be created. In addition to offer conversations, for a further relief a temporary sedated psychopharmacology can be necessary. In case of a suicide in a clinic, recommendations should be present that regulates responsibilities and procedures. Effective prevention methods are multiplier training, public education, restricted access to methods, and complying with media guidelines.

A novel subset of human regulatory B-cells has recently been described. They arise from within the transitional B-cell subpopulation and are characterised by the production of IL-10. They appear to be of significant importance in regulating T-cell immunity in vivo. Despite this important function, the molecular mechanisms by which they control T-cell activation are incompletely defined. Here we show that transitional B-cells produced more IL-10 and expressed higher levels of IL-10 receptor after CD40 engagement compared to other B-cell subsets. Furthermore, under this stimulatory condition, CD86 expressed by transitional B-cells was down regulated and T-cell proliferation was reduced. We provide evidence to demonstrate that the down-regulation of CD86 expression by transitional B-cells was due to the autocrine effect of IL-10, which in turn leads to decreased T-cell proliferation and TNF-α production. This analysis was further extended to peripheral B-cells in kidney transplant recipients. We observed that B-cells from patients tolerant to the graft maintained higher IL-10 production after CD40 ligation, which correlates with lower CD86 expression compared to patients with chronic rejection. Hence, the results obtained in this study shed light on a new alternative mechanism by which transitional B-cells inhibit T-cell proliferation and cytokine production.

Immunosuppressive drugs in clinical transplantation are necessary to inhibit the immune response to donor antigens. Although they are effective in controlling acute rejection, they do not prevent long-term transplant loss from chronic rejection. In addition, immunosuppressive drugs have adverse side effects, including increased rate of infections and malignancies. Adoptive cell therapy with human Tregs represents a promising strategy for the induction of transplantation tolerance. Phase I/II clinical trials in transplanted patients are already underway, involving the infusion of Tregs alongside concurrent immunosuppressive drugs. However, it remains to be determined whether the presence of immunosuppressive drugs negatively impacts Treg function and stability. We tested in vitro and in vivo the effects of tacrolimus, mycophenolate and methylprednisolone (major ISDs used in transplantation) on ex vivo expanded, rapamycin-treated human Tregs. The in vitro results showed that these drugs had no effect on phenotype, function and stability of Tregs, although tacrolimus affected the expression of chemokine receptors and IL-10 production. However, viability and proliferative capacity were reduced in a dose-dependent manner by all the three drugs. The in vivo experiments using a humanized mouse model confirmed the in vitro results. However, treatment of mice with only rapamycin maintained the viability, function and proliferative ability of adoptively transferred Tregs. Taken together, our results suggest that the key functions of ex vivo expanded Tregs are not affected by a concurrent immunosuppressive therapy. However, the choice of the drug combination and their timing and dosing should be considered as an essential component to induce and maintain tolerance by Treg., (Copyright© Ferrata Storti Foundation.)

Animal models have been instrumental in our understanding of the mechanisms of rejection and the testing of novel treatment options in the context of transplantation. We have now entered an exciting era with research on humanized mice driving advances in translational studies and in our understanding of the function of human cells in response to pathogens and cancer as well as the recognition of human allogeneic tissues in vivo. In this chapter we provide a historical overview of humanized mouse models of transplantation to date, outlining the distinct strains and share our experiences in the study of human transplantation immunology.

A simple dissolvable microneedle array (MA) platform has emerged as a promising technology for vaccine delivery, due to needle-free injection with a formulation that preserves the immunogenicity of live viral vectored vaccines dried in the MA matrix. While recent studies have focused largely on design parameters optimized to induce primary CD8(+) T cell responses, the hallmark of a vaccine is synonymous with engendering long-lasting memory. Here, we address the capacity of dried MA vaccination to programme phenotypic markers indicative of effector/memory CD8(+) T cell subsets and also responsiveness to recall antigen benchmarked against conventional intradermal (ID) injection. We show that despite a slightly lower frequency of dividing T cell receptor transgenic CD8(+) T cells in secondary lymphoid tissue at an early time point, the absolute number of CD8(+) T cells expressing an effector memory (CD62L(-)CD127(+)) and central memory (CD62L(+)CD127(+)) phenotype during peak expansion were comparable after MA and ID vaccination with a recombinant human adenovirus type 5 vector (AdHu5) encoding HIV-1 gag. Similarly, both vaccination routes generated CD8(+) memory T cell subsets detected in draining LNs for at least two years post-vaccination capable of responding to secondary antigen. These data suggest that CD8(+) T cell effector/memory generation and long-term memory is largely unaffected by physical differences in vaccine delivery to the skin via dried MA or ID suspension., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

B cells have been reported to promote graft rejection through alloantibody production. However, there is growing evidence that B cells can contribute to the maintenance of tolerance. Here, we used a mouse model of MHC-class I mismatched skin transplantation to investigate the contribution of B cells to graft survival. We demonstrate that adoptive transfer of B cells prolongs skin graft survival but only when the B cells were isolated from mice housed in low sterility "conventional" (CV) facilities and not from mice housed in pathogen free facilities (SPF). However, prolongation of skin graft survival was lost when B cells were isolated from IL-10 deficient mice housed in CV facilities. The suppressive function of B cells isolated from mice housed in CV facilities correlated with an anti-inflammatory environment and with the presence of a different gut microflora compared to mice maintained in SPF facilities. Treatment of mice in the CV facility with antibiotics abrogated the regulatory capacity of B cells. Finally, we identified transitional B cells isolated from CV facilities as possessing the regulatory function. These findings demonstrate that B cells, and in particular transitional B cells, can promote prolongation of graft survival, a function dependent on licensing by gut microflora.

Despite the availability of major histocompatibility complex (MHC)-binding peptide prediction algorithms, the development of T-cell vaccines against pathogen and tumor antigens remains challenged by inefficient identification of immunogenic epitopes. CD8(+) T cells must distinguish immunogenic epitopes from nonimmunogenic self peptides to respond effectively against an antigen without endangering the viability of the host. Because this discrimination is fundamental to our understanding of immune recognition and critical for rational vaccine design, we interrogated the biochemical properties of 9,888 MHC class I peptides. We identified a strong bias toward hydrophobic amino acids at T-cell receptor contact residues within immunogenic epitopes of MHC allomorphs, which permitted us to develop and train a hydrophobicity-based artificial neural network (ANN-Hydro) to predict immunogenic epitopes. The immunogenicity model was validated in a blinded in vivo overlapping epitope discovery study of 364 peptides from three HIV-1 Gag protein variants. Applying the ANN-Hydro model on existing peptide-MHC algorithms consistently reduced the number of candidate peptides across multiple antigens and may provide a correlate with immunodominance. Hydrophobicity of TCR contact residues is a hallmark of immunogenic epitopes and marks a step toward eliminating the need for empirical epitope testing for vaccine development.

Viral vectors are promising tools for vaccination strategies and immunotherapies. However, CD8⁺ T cell responses against pathogen-derived epitopes are usually limited to dominant epitopes and antibody responses to recombinant encoded antigens (Ags) are mostly weak. We have previously demonstrated that the timing of viral Ag expression in infected professional Ag-presenting cells strongly shapes the epitope immunodominance hierarchy. T cells recognizing determinants derived from late viral proteins have a clear disadvantage to proliferate during secondary responses. In this work we evaluate the effect of overexpressing the recombinant Ag using the modified vaccinia virus early/late promoter H5 (mPH5). Although the Ag-expression from the natural promoter 7.5 (P7.5) and the mPH5 seemed similar, detailed analysis showed that mPH5 not only induces higher expression levels than P7.5 during early phase of infection, but also Ag turnover is enhanced. The strong overexpression during the early phase leads to broader CD8 T cell responses, while preserving the priming efficiency of stable Ags. Moreover, the increase in Ag-secretion favors the induction of strong antibody responses. Our findings provide the rationale to develop new strategies for fine-tuning the responses elicited by recombinant modified vaccinia virus Ankara by using selected promoters to improve the performance of this viral vector.

Classic phylogenetic and modern population-based clustering methods were used to analyze hepatitis C virus (HCV) evolution in plasma and to assess viral compartmentalization within peripheral blood mononuclear cells (PBMCs) in 6 children during 3.2-9.6yr of follow-up. Population structure analysis of cloned amplicons encompassing hypervariable region 1 led to the distinction of two evolutionary patterns, one highly divergent and another one genetically homogeneous. Viral adaptability was reflected by co-evolution of viral communities switching rapidly from one to another in the context of divergence and stability associated with highly homogeneous communities which were replaced by new ones after long periods. Additionally, viral compartmentalization of HCV in PBMCs was statistically demonstrated, suggesting their role as a pool of genetic variability. Our results support the idea of a community-based structure of HCV viral populations during chronic infection and highlight a role of the PBMC compartment in the persistence of such structure., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8(+) T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c(+) dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c(+) MHCII(hi) CD8α(neg) epithelial cell adhesion molecule (EpCAM(neg)) CD11b(+) langerin (Lang; CD207)(neg) DCs, but neither Langerhans cells nor Lang(+) DCs were required for CD8(+) T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8(+) T-cell priming by live rAdHu5 MAs.

Unlabelled: Macrophages play an important role in the rejection of xenogeneic cells and therefore represent a major obstacle to generating chimeric mice with human xenografts that are useful tools for basic and preclinical medical research. The signal inhibitory regulatory protein α (SIRPα) receptor is a negative regulator of macrophage phagocytic activity and interacts in a species-specific fashion with its ligand CD47. Furthermore, SIRPα polymorphism in laboratory mouse strains significantly affects the extent of human CD47-mediated toleration of human xenotransplants. Aiming to minimize macrophage activity and thus optimize human cell engraftment in immunodeficient mice, we lentivirally transduced murine CD47 (Cd47) into human liver cells. Human HepG2 liver cells expressing Cd47 were less frequently contacted and phagocytosed by murine RAW264.7 macrophages in vitro than their Cd47-negative counterparts. For the generation of human-mouse chimeric livers in immunodeficient BALB-ΔRAG/γ(c) -uPA (urokinase-type plasminogen activator) mice, freshly thawed cryopreserved human hepatocytes were transduced with a lentiviral expression vector for Cd47 using a refined in vitro transduction protocol immediately before transplantation. In vivo, Cd47-positive human primary hepatocytes were selectively retained following engraftment in immunodeficient mice, leading to at least a doubling of liver repopulation efficiencies., Conclusion: We conclude that ectopic expression of murine Cd47 in human hepatocytes selectively favors engraftment upon transplantation into mice, a finding that should have a profound impact on the generation of robust humanized small animal models. Moreover, dominance of ectopically expressed murine Cd47 over endogenous human CD47 should also widen the spectrum of immunodeficient mouse strains suitable for humanization., (Copyright © 2012 American Association for the Study of Liver Diseases.)

There is an increasing body of evidence suggesting that the transfer of preformed MHC class I:peptide complexes between a virus-infected cell and an uninfected APC, termed cross-dressing, represents an important mechanism of Ag presentation to CD8+ T cells in host defense. However, although it has been shown that memory CD8+ T cells can be activated by uninfected dendritic cells (DCs) cross-dressed by Ag from virus-infected parenchymal cells, it is unknown whether conditions exist during virus infection in which naive CD8+ T cells are primed and differentiate to cytolytic effectors through cross-dressing, and indeed which DC subset would be responsible. In this study, we determine whether the transfer of MHC class I:peptide complexes between infected and uninfected murine DC plays a role in CD8+ T cell priming to viral Ags in vivo. We show that MHC class I:peptide complexes from peptide-pulsed or virus-infected DCs are indeed acquired by splenic CD8α⁻ DCs in vivo. Furthermore, the acquired MHC class I:peptide complexes are functional in that they induced Ag-specific CD8+ T cell effectors with cytolytic function. As CD8α⁻ DCs are poor cross-presenters, this may represent the main mechanism by which CD8α⁻ DCs present exogenously encountered Ag to CD8+ T cells. The sharing of Ag as preformed MHC class I:peptide complexes between infected and uninfected DCs without the restraints of Ag processing may have evolved to accurately amplify the response and also engage multiple DC subsets critical in the generation of strong antiviral immunity.

The implementation of vaccination as an empiric strategy to protect against infectious diseases was introduced even before the advent of hygiene and antimicrobials in the medical practice. Nevertheless, it was not until a few decades ago that we really started understanding the underlying mechanisms of protection triggered by vaccination. Vaccines were initially based on attenuated or inactivated organisms. Subunit vaccines were then introduced as more refined formulations, exhibiting improved safety profiles. However, purified antigens tend to be poorly immunogenic and often require the use of adjuvants to achieve adequate stimulation of the immune system. Vaccination strategies, such as mucosal administration, also require potent adjuvants to improve performance. In the 1990s, immunologists found that pathogens could be sensed as 'danger signals' by receptors recognizing conserved motifs. Although our knowledge is still limited, tremendous advances were made in the understanding of host defence mechanisms regulated by these evolutionary conserved receptors, and the molecular structures which are recognized by them. This opened a new era in adjuvant development. Some of the latest players arrived to this field are the cyclic di-nucleotides, which are ubiquitous prokaryotic intracellular signalling molecules. This review is focused on their potential for the development of vaccines and immunotherapies., (© 2011 The Authors. Microbial Biotechnology © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Cytokine immunotherapies targeting T lymphocytes are attractive clinical interventions against viruses and tumors. In the mouse, the homeostasis of memory α/β CD8(+) T cells and natural killer (NK) cells is significantly improved with increased IL-15 bioavailability. In contrast, the role of "transpresented" IL-15 on human T-cell development and homeostasis in vivo is unknown. We found that both CD8 and CD4 T cells in human immune system (HIS) mice are highly sensitive to transpresented IL-15 in vivo, with both naïve (CD62L(+)CD45RA(+)) and memory phenotype (CD62L(-)CD45RO(+)) subsets being significantly increased following IL-15 "boosting." The unexpected global improvement in human T-cell homeostasis involved enhanced proliferation and survival of both naïve and memory phenotype peripheral T cells, which potentiated B-cell responses by increasing the frequency of antigen-specific responses following immunization. Transpresented IL-15 did not modify T-cell activation patterns or alter the global T-cell receptor (TCR) repertoire diversity. Our results indicate an unexpected effect of IL-15 on human T cells in vivo, in particular on CD4(+) T cells. As IL-15 promotes human peripheral T-cell homeostasis and increases the frequency of neutralizing antibody responses in HIS mice, IL-15 immunotherapy could be envisaged as a unique approach to improve vaccine responses in the clinical setting.

Background: Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the 'humanization' of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique., Methodology/principal Findings: After transplantation with CD34+CD38⁻ human hematopoietic progenitor cells, BALB/c Rag2⁻/⁻IL-2Rγc⁻/⁻ mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. "Human Immune System" mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19+CD27+ B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively., Conclusion/significance: This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens.

Expression vectors appear to be an indispensable tool for both biological studies and biotechnological applications. Controlling gene overexpression becomes a critical issue when protein production is desired. In addition to several aspects regarding toxicity or plasmid instability, tight control of gene expression is an essential factor in biotechnological processes. Thus, the search for better-controlled circuits is an important issue among biotechnologists. Traditionally, expression systems involve a single regulatory protein operating over a target promoter. However, these circuits are limited on their induction ratios (e.g., by their restriction in the maximal expression capacity, by their leakiness under non-induced conditions). Due to these limitations, regulatory cascades, which are far more efficient, are necessary for biotechnological applications. Thus, regulatory circuits with two modules operating in cascade offer a significant advantage. In this review, we describe the regulatory cascade based on two salicylate-responsive transcriptional regulators of Pseudomonas putida (nahR/P(sal)::xylS2), its properties, and contribution to a tighter control over heterologous gene expression in different applications.Nowadays, heterologous expression has been proven to be an indispensable tool for tackling basic biological questions, as well as for developing biotechnological applications. As the nature of the protein of interest becomes more complex, biotechnologists find that a tight control of gene expression is a key factor which conditions the success of the downstream purification process, as well as the interpretation of the results in other type of studies. Fortunately, different expression systems can be found in the market, each of them with their own pros and cons. In this review we discuss the exploitation of prokaryotic expression systems based on a promising expression system, the salicylate-dependent control circuit encompassing nahR/P(sal)::xylS2, as well as some of the improvements that have been done on this system to exploit it more efficiently in the context of both biotechnological applications and basic research., (© 2010 Landes Bioscience)

Despite the strong immune responses elicited after natural infection with Trypanosoma cruzi or vaccination against it, parasite survival suggests that these responses are insufficient or inherently inadequate. T. cruzi contains a major cystein proteinase, cruzipain, which has a catalytic N-terminal domain and a C-terminal extension. Immunizations that employed recombinant cruzipain or its N- and C-terminal domains allowed evaluation of the ability of cruzipain to circumvent responses against the catalytic domain. This phenomenon is not a property of the parasite but of cruzipain itself, because recombinant cruzipain triggers a response similar to that of cruzipain during natural or experimental infection. Cruzipain is not the only antigen with a highly immunogenic region of unknown function that somehow protects an essential domain for parasite survival. However, our studies show that this can be reverted by using the N-terminal domain as a tailored immunogen able to redirect host responses to provide enhanced protection.

Background: Modified vaccinia virus Ankara (MVA), a highly attenuated strain of vaccinia virus, has been used as vaccine delivery vector in preclinical and clinical studies against infectious diseases and malignancies. Here, we investigated whether an MVA which does not encode any antigen (Ag) could be exploited as adjuvant per se., Methodology/principal Findings: We showed that dendritic cells infected in vitro with non-recombinant (nr) MVA expressed maturation and activation markers and were able to efficiently present exogenously pulsed Ag to T cells. In contrast to the dominant T helper (Th) 1 biased responses elicited against Ags produced by recombinant MVA vectors, the use of nrMVA as adjuvant for the co-administered soluble Ags resulted in a long lasting mixed Th1/Th2 responses., Conclusions/significance: These findings open new ways to potentiate and modulate the immune responses to vaccine Ags depending on whether they are co-administered with MVA or encoded by recombinant viruses.

Mucosal dendritic cells (DCs) are very important in the process of antigen presentation to T cells, playing a key role in the induction of primary and secondary immune responses. Pidotimod is a synthetic substance capable of modulating immune cell functions, but the effect of pidotimod on human DCs has not been investigated yet. Here we demonstrate the ability of pidotimod to induce DC maturation and up-regulate the expression of HLA-DR and co-stimulatory molecules CD83 and CD86, which are fundamental for communication with adaptative immunity cells. Pidotimod also stimulated DCs to release high amounts of pro-inflammatory molecules such as MCP-1 and TNF-alpha cytokines and to drive T cell proliferation and differentiation towards a Th1 phenotype. Moreover, we demonstrate that pidotimod in vivo promotes strong and specific humoral and cellular immune response when co-administered intranasally with a model antigen. Taken together our data suggest the possibility to use pidotimod as adjuvant molecule to facilitate the activation of the innate immune system as well as to promote an effective mucosal and systemic immune response.

Fetal liver progenitor cell suspensions (FLPC) and hepatic precursor cells derived from embryonic stem cells (ES-HPC) represent a potential source for liver cell therapy. However, the relative capacity of these cell types to engraft and repopulate a recipient liver compared with adult hepatocytes (HC) has not been comprehensively assessed. We transplanted mouse and human HC, FLPC, and ES-HPC into a new immunodeficient mouse strain (Alb-uPA(tg(+/-))Rag2(-/-)gamma(c)(-/-) mice) and estimated the percentages of HC after 3 months. Adult mouse HC repopulated approximately half of the liver mass (46.6 +/- 8.0%, 1 x 10(6) transplanted cells), whereas mouse FLPC derived from day 13.5 and 11.5 post conception embryos generated only 12.1 +/- 3.0% and 5.1 +/- 1.1%, respectively, of the recipient liver and smaller cell clusters. Adult human HC and FLPC generated overall less liver tissue than mouse cells and repopulated 10.0 +/- 3.9% and 2.7 +/- 1.1% of the recipient livers, respectively. Mouse and human ES-HPC did not generate HC clusters in our animal model. We conclude that, in contrast to expectations, adult HC of human and mouse origin generate liver tissue more efficiently than cells derived from fetal tissue or embryonic stem cells in a highly immunodeficient Alb-uPA transgenic mouse model system. These results have important implications in the context of selecting the optimal strategy for human liver cell therapies.

Over 800 million people worldwide are infected with hepatitis viruses, human immunodeficiency virus (HIV), and malaria, resulting in more than 5 million deaths annually. Here we discuss the potential and challenges of humanized mouse models for developing effective and affordable therapies and vaccines, which are desperately needed to combat these diseases.

Hepatitis C virus (HCV) hypervariable region 1 (HVR1) is the most variable region of the viral genome and its heterogeneity reflects the virus-host interplay during chronicity. Paediatric HCV-infected patients develop liver disease with typical clinical features. Here, the evolution of HVR1 and its adjacent regions were ascertained in plasma samples of two HCV-positive children during a 5-year follow-up period. We report an almost complete conservation of the HVR1 amino acid sequence over time, with underlying nucleotide variability both within and outside HVR1, suggesting some kind of constraint on virus evolution, particularly within HVR1. Although overall d(N)/d(S) rates [rates of nonsynonymous nucleotide substitutions per nonsynonymous site (d(N)) and synonymous nucleotide substitutions per synonymous site (d(S))] were <1 in both patients, a high resolution analysis of selection pressures exerted at the codon level revealed few sites subject to selection and an absolute predominance of invariable positions within HVR1. The HVR1 amino acid sequences showed the antigenic properties expected for this region. Taken together, these data suggest peculiar evolutionary dynamics in our patients, which could be attributed to a mechanism of nucleotide invariability along with purifying selection operating on the HVR1. The lack of HVR1 variability may reflect the adaptation of the virus to a particular environment within each patient or a phenomenon of immune tolerance generated in these immunocompetent patients earlier in life.

We recently showed that treatment of macrophages prior to Mycobacterium tuberculosis infection with the pro-inflammatory omega-6 lipid, arachidonic acid (AA) enhanced bacterial killing whereas the anti-inflammatory, omega-3 lipid eicosapentaenoic acid (EPA) stimulated bacterial growth. Here we tested if these effects were depending on when lipids were added to macrophages: before or during Mycobacterium smegmatis or M. tuberculosis infection. Collectively, our data suggested that a high omega-6 diet might be beneficial against mycobacteriosis, while a high omega-3 diet might be detrimental. AA also stimulated TNF-alpha secretion in M. tuberculosis-infected macrophages whereas EPA inhibited this process. AA strongly activated the MAP kinase p38 in uninfected cells but M. tuberculosis infected cells blocked the ability of AA to activate p38; AA-dependent killing is therefore independent of p38. We therefore tested diets enriched in omega-3 and omega-6 lipids on a mouse model of tuberculosis. In contrast to the in vitro results, the omega-6 tended to increase survival of M. tuberculosis in mice, while omega-3- tended to increase pathogen killing. Overall our results together with those previously reported in the literature suggest that it is almost impossible to predict, at the whole organism level, if a diet enriched in omega-3 or -6 will be beneficial or detrimental to intracellular pathogens.

The major challenge for the development of a highly effective peptide-based vaccine is represented by the diversity of HIV-1 strains among human population. HIV-1 matrix protein p17 is a candidate antigen for therapeutic vaccines against AIDS. Here we show that antibodies elicited in animals by immunizing them with a synthetic peptide representative of the p17 functional epitope (AT20) derived from HIV-1 BH10 (clade B), neutralize the biological activity of p17 derived from divergent strains displaying critical mutations within AT20, by recognizing a highly conserved conformational epitope. This finding shows that AT20, as an immunogenic molecule, elicits broadly neutralizing anti-p17 antibodies.

The so-called DNA vaccination represents one of the most notable tools under development in the field of vaccinology. The concept of administering the gene coding for any given protective antigen and make responsible vaccinee's own cells to produce the protein appeals as too simple to be true. Indeed, the implementation of this approach for mass vaccination should overcome several bottlenecks, such as need of high dosages and poor immunogenicity. In this context, the use of live attenuated bacteria as delivery system for plasmid DNA has emerged as a promising alternative to overcome many of those pitfalls. In addition, this approach is not only amenable for mucosal administration, but allows to specifically target professional antigen presenting cells. This results in their transfection, as well as in their activation and maturation, due to their built-in adjuvant properties resulting from the stimulation of pattern recognition receptors. This chapter discusses the specific features that should be taken into consideration when designing a plasmid vector, current candidate bacterial carriers for DNA delivery and main safety issues.

CpG are powerful drugs activating the innate immune system. In this study, the ability of their intramammary administration in impeding the devastating progression of carcinogenesis in all the mammary glands of female BALB/c mice transgenic for the rat neu transforming oncogene was assessed. Starting when in situ carcinomas were scattered over all their mammary glands (week 10), mice received CpG injections in the stroma of the fourth left gland. Local neoplastic progression was inhibited by six monthly administrations. CpG not only delayed the onset of carcinomas in the injected gland, but also hampered their progression. Extended latency was observed for tumors in glands both close to and far from the injection site. When the experiment ended (week 45), no tumors were palpable in 67% of the injected glands and a markedly impaired tumor growth was evident in the others. An impressive local infiltrate of CD11b(+) cells with the morphologic features of macrophages, plasma cells, B220(+) B cells, and CD4(+) and CD8(+) T cells was quickly recruited to the CpG-treated glands. High quantities of IFN-gamma producing cells were only present in the ipsilateral axillary draining lymph nodes of the treated glands. Enhanced natural killer (NK) lytic activity was also detected in the spleens. Inhibition of progression was weaker when only four injections were given, and abolished by in vivo depletion of NK cells. CpG monotherapy is thus effective in an aggressive model of autochthonous cancer. The results strongly support the administration of CpG as a local monotherapy of multiple invasive microscopic lesions.

Cruzipain (Cz), a key Trypanosoma cruzi enzyme, is a main candidate antigen for vaccines against Chagas' disease. We evaluated a vaccination protocol based on intradermal priming with recombinant Cz and intranasal boosting with rCz co-administered with a derivative of the TLR2/6 agonist MALP-2. Vaccination triggered strong systemic and mucosal antibody responses, and a vigorous cell-mediated immunity characterized by lymphoproliferation, DTH reactivity and IFN-gamma production. The immune responses protected against a lethal trypomastigote challenge and, upon sub-lethal infection, immunized mice showed reduction of tissue damage and normal enzymatic markers of muscle injury. This prime-boost regimen appears promising for further development, since warranted survival, provided efficient control of parasite load and restricted inflammatory myopathy.

Numerical and functional defects in plasmacytoid dendritic cells (pDCs) are an important hallmark of progressive HIV-1 infection, yet its etiology remains obscure. HIV-1 p17 matrix protein (p17) modulates a variety of cellular responses, and its biological activity depends on the expression of p17 receptors (p17Rs) on the surface of target cells. In this study, we show that peripheral blood pDCs express p17Rs on their surface and that freshly isolated pDCs are sensitive to p17 stimulation. Upon p17 treatment, pDCs undergo phenotypic differentiation with up-regulation of CCR7. A chemotaxis assay reveals that p17-treated pDCs migrate in response to CCL19, suggesting that these cells may acquire the ability to migrate to secondary lymphoid organs. In contrast, p17 does not induce release of type I IFN nor does it enhance pDC expression of CD80, CD86, CD83, or MHC class II. Microarray gene expression analysis indicated that p17-stimulated pDCs down-regulate the expression of molecules whose functions are crucial for efficient protein synthesis, protection from apoptosis, and cell proliferation induction. Based on these results, we propose a model where p17 induces immature circulating pDCs to home in lymph nodes devoid of their ability to serve as a link between innate and adaptative immune systems.

To stimulate both local and systemic immune responses against Trypanosoma cruzi, Salmonella enterica serovar Typhimurium aroA was exploited as a DNA delivery system for cruzipain (SCz). In a murine model we compared SCz alone (GI) or coadministered with Salmonella carrying a plasmid encoding granulocyte-macrophage colony-stimulating factor (GII), as well as protocols in which SCz priming was followed by boosting with recombinant cruzipain (rCz) admixed with either CpG-ODN (GIII) or MALP-2, a synthetic derivative of a macrophage-activating lipopeptide of 2 kDa from Mycoplasma fermentans (GIV). The results showed that protocols that included four oral doses of SCz (GI) elicited mainly a mucosal response characterized by immunoglobulin A (IgA) secretion and proliferation of gut-associated lymphoid tissue cells, with weak systemic responses. In contrast, the protocol that included a boost with rCz plus CpG (GIII) triggered stronger systemic responses in terms of Cz-specific serum IgG titers, splenocyte proliferation, gamma interferon (IFN-gamma) secretion, and delayed-type hypersensitivity response. Trypomastigote challenge of vaccinated mice resulted in significantly lower levels of parasitemia compared to controls. Protection was abolished by depletion of either CD4+ or CD8+ T cells. Parasite control was also evident from the reduction of tissue damage, as revealed by histopathologic studies and serum levels of enzymes that are markers of muscle injury in chronic Chagas' disease (i.e., creatine kinase, aspartate aminotransferase, and lactate dehydrogenase). Enhanced release of IFN-gamma and interleukin-2 was observed in GI and GII upon restimulation of splenocytes in the nonparasitic phase of infection. Our results indicate that Salmonella-mediated delivery of Cz-DNA by itself promotes the elicitation of an immune response that controls T. cruzi infection, thereby reducing parasite loads and subsequent damage to muscle tissues.

Systems allowing tightly regulated expression of prokaryotic genes in vivo are important for performing functional studies of bacterial genes in host-pathogen interactions and establishing bacteria-based therapies. We integrated a regulatory control circuit activated by acetyl salicylic acid (ASA) in attenuated Salmonella enterica that carries an expression module with a gene of interest under control of the XylS2-dependent Pm promoter. This resulted in 20-150-fold induction ex vivo. The regulatory circuit was also efficiently induced by ASA when the bacteria resided in eukaryotic cells, both in vitro and in vivo. To validate the circuit, we administered Salmonella spp., carrying an expression module encoding the 5-fluorocytosine-converting enzyme cytosine deaminase in the bacterial chromosome or in a plasmid, to mice with tumors. Induction with ASA before 5-fluorocytosine administration resulted in a significant reduction of tumor growth. These results demonstrate the usefulness of the regulatory control circuit to selectively switch on gene expression during bacterial infection.

Both neutralizing antibodies and cytotoxic T-cells are necessary to control a viral infection. However, vigorous T helper responses are essential for their elicitation and maintenance. Here we show that a recombinant replication-deficient Herpes Simplex Virus (HSV)-1 vector encoding the Human Immunodeficiency Virus (HIV)-1 matrix protein p17 (T0-p17) was capable of infecting professional antigen presenting cells (APCs) in vitro and in vivo. The injection of T0-p17 in the mouse dermis generated a strong p17-specific CD4+ T helper response preceding both p17-specific humoral and effector T cell responses. Moreover, we show that T0-p17 infection did not interfere with the endogenous processing of the transgene encoded antigen, since infected APCs were able to evoke a strong recall response in vitro. Our results demonstrate that replication-deficient HSV vectors can be appealing candidates for the development of vaccines able to trigger T helper responses.

Adamantylamide L-alanyl-D-isoglutamine (AdDP) is a synthetic adjuvant which belongs to the family of the desmuramyl peptides. AdDP exerts its adjuvant properties when it is administered either by the parenteral or by the mucosal route, leading to the elicitation of strong humoral responses at both the systemic and the mucosal levels. However, very little is known about the effect of AdDP on cellular immunity. Here we demonstrate that AdDP is able to stimulate cellular responses, which are characterized by the release of gamma interferon by CD8+ T cells when they are restimulated with a major histocompatibility complex class I-restricted peptide and strong in vivo lymphocyte-mediated cytotoxic activity. The capacity of AdDP to stimulate the elicitation of both cellular and humoral adaptive responses makes this adjuvant a promising tool for the development of mucosal vaccine formulations.

Moraxella catarrhalis causes acute otitis media in children and lower respiratory tract infections in adults and elderly. In children the presence of antibodies against the highly conserved outer membrane protein CD correlates with protection against infection, suggesting that this protein may be useful as a vaccine antigen. However, native CD is difficult to purify, and it is still unclear if recombinant CD (rCD) is a valid alternative. We performed a side-by-side comparison of the immunogenicities and efficacies of vaccine formulations containing native CD and rCD with adamantylamide dipeptide as the mucosal adjuvant. Intranasal vaccination of mice stimulated the production of high CD-specific antibody titers in sera and of secretory immunoglobulin A in mucosal lavages, which cross-recognized both antigens. While vaccination with native CD increased the number of interleukin-2 (IL-2)- and gamma interferon-producing cells, rCD mainly stimulated IL-4-secreting cells. Nevertheless, efficient bacterial clearance was observed in the lungs of challenged mice receiving native CD and in the lungs of challenged mice receiving rCD (96% and 99%, respectively). Thus, rCD is a promising candidate for incorporation in vaccine formulations for use against M. catarrhalis.

The HIV-1 matrix protein p17 is a structural protein essential in the life cycle of HIV, by acting as a virokine/immunomodulator that supports viral replication and spreading. The presence of p17-specific antibodies and CTL responses correlates with slower progression to AIDS. Intranasal vaccination with p17 and the TLR2/6 agonist MALP-2 stimulates strong humoral and cellular immune responses at systemic and mucosal levels. The antibodies blocked p17 binding to its receptor, which is a critical step for the exertion of its virokine activity. Our results suggest that p17 and MALP-2 are attractive candidates for incorporation in mucosal vaccines against HIV/AIDS.

HIV-1 p17 is a viral cytokine that acts on preactivated, but not on resting, human T cells promoting proliferation, proinflammatory cytokines release and HIV-1 replication, after binding to a cellular receptor (p17R). Here, we demonstrate that p17Rs are expressed on activated murine T cells, which respond to p17 stimulation similarly to their human counterpart. We developed a mouse model of abortive HSV-1 infection to induce T cell activation in vivo. Preactivated cells expressed p17Rs and were highly susceptible to p17 stimulation, which triggered proinflammatory cytokines release and promoted CD4+ T cell survival and expansion. Coculture of in vivo activated splenocytes with macrophages in the presence of p17 further increased their ability to produce IFN-gamma. The presence of macrophages and activated T cells at mucosal sites prompted us to investigate the immunomodulatory activities of p17 in vivo. Intranasal coadministration of p17 with beta-galactosidase (beta-gal) resulted in improved beta-gal specific cellular and humoral immune responses at systemic and mucosal levels. It is well established that HIV-1 replication is driven in an autocrine/paracrine manner by endogenously produced proinflammatory cytokines. Our results highlight the role of p17 in sustaining cellular activation and inflammation, thereby promoting a permissive microenvironment for HIV-1 replication. In addition, p17 is a promising candidate antigen, exhibiting immunomodulatory/adjuvant properties, that need to be exploited in the development of HIV/AIDS vaccines.