Michael Palm Basic Science, Vaccines & Prevention Project Weblog

Trends in Immunology: Free Access Issue on Immune Senescence

The journal Trends in Immunology has published a special issue on immune senescence, and access to all articles is being offered free of charge (thanks to the sponsorship of the National Institutes of Health). The parallels between immune senescence in uninfected elderly individuals and people with HIV are increasingly well-recognized and include:

• Depletion of naive CD4 and CD8 T cells
• Involution of the thymus and decreased thymic output
• Skewing of the CD4:CD8 T cell ratio
• Accumulation of dysfunctional CD8 T cells lacking the CD28 co-stimulatory molecule
• Decreased responses to new immunizations
• Decreased recall (memory) T responses to common antigens

Research into immune senescence and its association with illness and mortality has gained considerably more attention recently, and may have the potential to offer important insights into the pathogenesis of HIV infection. Additional background on the topic, along with a link to a webcast presentation by immune senescence expert Rita Effros, can be found in a post from January of this year, "Accelerated Aging of the Immune System in HIV Infection."

Short Cuts To Memory

Two papers in last week’s issue of Nature report that memory CD8 T cell responses can be enhanced by drugs that alter cellular metabolism. In one of the studies, the drug rapamycin – generally used as an immune suppressant – also accelerated the development of functional memory CD8 T cells. The findings may assist efforts to develop more effective CD8 T cell vaccines and therapeutics, because one of the inconvenient aspects of T cell biology is that the differentiation process by which naïve T cells become fully functional memory T cells takes time (several weeks in mice and considerably longer in people). Furthermore, restimulation of the T cells during this differentiation process can be counterproductive, reducing the quality of the resultant memory response.

On a somewhat similar theme, the new issue of Nature Medicine includes a paper showing that manipulation of the Wnt signaling pathway can also arrest the normal T cell differentiation process, leading to the generation of memory CD8 T cells with stem cell-like self-renewal properties.

Nature 460, 41-42 (2 July 2009) | doi:10.1038/460041a; Published online 1 July 2009

News and Views

Immunology: A metabolic switch to memory

Martin Prlic & Michael J. Bevan

Abstract

Two therapeutic drugs have been found to enhance memory in immune cells called T cells, apparently by altering cellular metabolism. Are changes in T-cell metabolism the key to generating long-lived immune memory?

Nature 460, 103-107 (2 July 2009) | doi:10.1038/nature08097; Received 23 January 2009; Accepted 23 April 2009; Published online 3 June 2009

Enhancing CD8 T-cell memory by modulating fatty acid metabolism

Erika L. Pearce1, Matthew C. Walsh1, Pedro J. Cejas1, Gretchen M. Harms1, Hao Shen2, Li-San Wang1,3, Russell G. Jones4 & Yongwon Choi1

1. Department of Pathology and Laboratory Medicine,

2. Department of Microbiology,

3. Penn Center for Bioinformatics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA

4. McGill Cancer Centre, Department of Physiology, McGill University, Montreal, QC, H3G 1Y6, Canada

CD8 T cells, which have a crucial role in immunity to infection and cancer, are maintained in constant numbers, but on antigen stimulation undergo a developmental program characterized by distinct phases encompassing the expansion and then contraction of antigen-specific effector (TE) populations, followed by the persistence of long-lived memory (TM) cells Although this predictable pattern of CD8 T-cell responses is well established, the underlying cellular mechanisms regulating the transition to TM cells remain undefined. Here we show that tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), an adaptor protein in the TNF-receptor and interleukin-1R/Toll-like receptor superfamily, regulates CD8 TM-cell development after infection by modulating fatty acid metabolism. We show that mice with a T-cell-specific deletion of TRAF6 mount robust CD8 TE-cell responses, but have a profound defect in their ability to generate TM cells that is characterized by the disappearance of antigen-specific cells in the weeks after primary immunization. Microarray analyses revealed that TRAF6-deficient CD8 T cells exhibit altered expression of genes that regulate fatty acid metabolism. Consistent with this, activated CD8 T cells lacking TRAF6 display defective AMP-activated kinase activation and mitochondrial fatty acid oxidation (FAO) in response to growth factor withdrawal. Administration of the anti-diabetic drug metformin restored FAO and CD8 TM-cell generation in the absence of TRAF6. This treatment also increased CD8 TM cells in wild-type mice, and consequently was able to considerably improve the efficacy of an experimental anti-cancer vaccine.

Nature 460, 108-112 (2 July 2009) | doi:10.1038/nature08155; Received 27 April 2009; Accepted 15 May 2009; Published online 21 June 2009

mTOR regulates memory CD8 T-cell differentiation

Koichi Araki1, Alexandra P. Turner2, Virginia Oliva Shaffer2, Shivaprakash Gangappa2, Susanne A. Keller3, Martin F. Bachmann3, Christian P. Larsen2 & Rafi Ahmed1

1. Emory Vaccine Center and Department of Microbiology and Immunology,

2. Emory Transplant Center and Department of Surgery, Emory University School of Medicine, Atlanta, Georgia 30322, USA

3. Cytos Biotechnology AG, Wagistrasse 25, 8952 Zürich-Schlieren, Switzerland

Memory CD8 T cells are a critical component of protective immunity, and inducing effective memory T-cell responses is a major goal of vaccines against chronic infections and tumours. Considerable effort has gone into designing vaccine regimens that will increase the magnitude of the memory response, but there has been minimal emphasis on developing strategies to improve the functional qualities of memory T cells. Here we show that mTOR (mammalian target of rapamycin, also known as FRAP1) is a major regulator of memory CD8 T-cell differentiation, and in contrast to what we expected, the immunosuppressive drug rapamycin has immunostimulatory effects on the generation of memory CD8 T cells. Treatment of mice with rapamycin following acute lymphocytic choriomeningitis virus infection enhanced not only the quantity but also the quality of virus-specific CD8 T cells. Similar effects were seen after immunization of mice with a vaccine based on non-replicating virus-like particles. In addition, rapamycin treatment also enhanced memory T-cell responses in non-human primates following vaccination with modified vaccinia virus Ankara. Rapamycin was effective during both the expansion and contraction phases of the T-cell response; during the expansion phase it increased the number of memory precursors, and during the contraction phase (effector to memory transition) it accelerated the memory T-cell differentiation program. Experiments using RNA interference to inhibit expression of mTOR, raptor (also known as 4932417H02Rik) or FKBP12 (also known as FKBP1A) in antigen-specific CD8 T cells showed that mTOR acts intrinsically through the mTORC1 (mTOR complex 1) pathway to regulate memory T-cell differentiation. Thus these studies identify a molecular pathway regulating memory formation and provide an effective strategy for improving the functional qualities of vaccine- or infection-induced memory T cells.

Nature Medicine 15, 731 - 732 (2009), doi:10.1038/nm0709-731

Tumor immunotherapy: making an immortal army

Brent H. Koehn1 & Stephen P. Schoenberger1

Laboratory of Cellular Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA.

Abstract

Manipulation of cell renewal pathways creates T memory stem cells that can generate a sustained and targeted immune response. These findings have broad implications for vaccine development and immunotherapy (pages 808–813).

Nature Medicine 15, 808 - 813 (2009)

Published online: 14 June 2009 | doi:10.1038/nm.1982

Wnt signaling arrests effector T cell differentiation and generates CD8+ memory stem cells

Luca Gattinoni1,2, Xiao-Song Zhong1,2, Douglas C Palmer1, Yun Ji1, Christian S Hinrichs1, Zhiya Yu1, Claudia Wrzesinski1, Andrea Boni1, Lydie Cassard1, Lindsay M Garvin1, Chrystal M Paulos1, Pawel Muranski1 & Nicholas P Restifo1

1. Center for Cancer Research, National Cancer Institute, US National Institutes of Health, Bethesda, Maryland, USA.

2. These authors contributed equally to this work.

Self-renewing cell populations such as hematopoietic stem cells and memory B and T lymphocytes might be regulated by shared signaling pathways1. The Wnt–-catenin pathway is an evolutionarily conserved pathway that promotes hematopoietic stem cell self-renewal and multipotency by limiting stem cell proliferation and differentiation2, 3, but its role in the generation and maintenance of memory T cells is unknown. We found that induction of Wnt–-catenin signaling by inhibitors of glycogen sythase kinase-3 or the Wnt protein family member Wnt3a arrested CD8+ T cell development into effector cells. By blocking T cell differentiation, Wnt signaling promoted the generation of CD44lowCD62LhighSca-1highCD122highBcl-2high self-renewing multipotent CD8+ memory stem cells with proliferative and antitumor capacities exceeding those of central and effector memory T cell subsets. These findings reveal a key role for Wnt signaling in the maintenance of 'stemness' in mature memory CD8+ T cells and have major implications for the design of new vaccination strategies and adoptive immunotherapies.

Memory in the Bones

A new paper in the journal Immunity reports that a substantial proportion of antigen-specific memory CD4 T cells take up residence in the bone marrow after an immune response, at least in mice. The paper adds to a growing body of evidence that the bone marrow represents a key – perhaps the key – site for the maintenance of immunological memory. It was established by Mark Slifka in 1995 that after both a primary and secondary infection with LCMV, plasma B cells preferentially migrate to the bone marrow and sustain antiviral antibody production there. More recently, it has been shown that the bone marrow also contains a major reservoir of central memory CD8 T cells and is the main site of their homeostatic proliferation.

But the picture for memory CD4 T cells has been less clear. As far back as 1974 it was reported that mouse CD4 T cells migrate to the bone marrow after priming but it wasn’t until over twenty years later that a study formally demonstrated that tetanus-specific memory CD4 T cells can be transferred from donor to recipient during bone marrow transplantation (with the number of cells increasing if the donor was immunized prior to donation). Very recently, Mirko Paiardini and colleagues from Guido Silvestri’s laboratory published a paper in Blood showing that the bone marrow supports extensive homeostatic proliferation of both memory CD4 and CD8 T cells in non-human primates. 

In HIV-infected humans, there is an interesting radiolabeling study from 2002, which reported that a majority of CD4 T cells labeled ex vivo and reinfused trafficked to the bone marrow. The rate of migration was greater in HIV-infected participants compared to uninfected controls: “Another obvious finding is the increased intensity of labeled CD4 T cells in the vertebral and iliac bone marrow… The two other HIV+ subjects tested displayed similar enhanced accumulation of CD4 T cells in the bone, compared with the two other control subjects. It was also obvious that most of the labeled CD4 T cells in normal subjects migrated to bone marrow, and this was almost exclusively to vertebral and iliac marrow and not marrow in the long bones in both types of subjects.” However, literature addressing whether HIV-infected memory CD4 T cells can be found in the bone marrow appears sparse to non-existent – I tried searching PubMed without success. It is tempting to wonder if there may be some connection with the isolated reports that have occurred regarding bone marrow transplantation and putative eradication of HIV.

But to get back to the details of the new Immunity paper, a group of scientists led by Koji Tokoyoda studied CD4 responses to a variety of antigens (LCMV, ovalbumin & KLH) and report that as memory develops, the number of antigen-specific CD4 T cells in the spleen and lymph nodes declines, while numbers in the bone marrow increase (a finding that very much echoes Mark Slifka's seminal data on plasma B cells). From day 60 after antigen challenge, the majority of antigen-specific memory CD4 T cells (~80%) were found in the bone marrow and numbers remained relatively stable out to more than 134 days. Additional analyses showed that almost all of these cells were resting based on DNA content and gene expression, and they appeared to be in contact with IL-7-expressing stromal cells. The researchers also show that memory CD4 T cells from the bone marrow can support the generation of antibodies by B cells, although they state that their data suggests that this involves the migration of the cells out of the bone marrow to secondary lymphoid tissues.

The study authors argue that their data will require the revision of prior models of how memory CD4 T cells are maintained (the generally accepted model is that most memory CD4 T cells constantly recirculate while surveilling for antigens); they propose a new division of “resting versus mobilized memory.” However, there are some confusing aspects of the study (although they may only be confusing to people not steeped in murine immunology). In the discussion section at the end, it’s stated that: “central memory T cells expressing CCR7 and CD62L were not found in the bone marrow,” but no data is presented in support of the statement. Yet central memory CD4 T cells are considered to be a very important pool of memory CD4 T cells as they generate effector cells upon re-exposure to antigen (the pool of central memory CD4 T cells is also proportionally larger in humans than it is in mice). Also, in the case of CD8 T cells, it is the central memory subset that preferentially traffics through, and proliferates in, the bone marrow. Prior studies have also universally characterized bone marrow CD4 T cells as being “activated” based on phenotypic criteria, and it would have been perhaps useful for Tokoyoda and colleagues to offer more discussion of how their study may be reconciled with the existing literature.

While there are clearly many questions yet to be addressed, the emergence of the bone marrow’s role in immunological memory represents an important new front in immunology research. For additional information, there is an excellent and extensive review of the subject by Francesca Di Rosa available online in Immunology and Cell Biology (access is free of charge).

Immunity, 07 May 2009

doi:10.1016/j.immuni.2009.03.015

Professional Memory CD4+ T Lymphocytes Preferentially Reside and Rest in the Bone Marrow

Koji Tokoyoda1, Sandra Zehentmeier1, Ahmed N. Hegazy1,2, Inka Albrecht1,  Joachim R. Grün1, Max Löhning1,2 and Andreas Radbruch1

1 Deutsches Rheuma-Forschungszentrum (DRFZ), Charitéplatz 1, Berlin 10117, Germany

2 Experimental Immunology, Department of Rheumatology and Clinical Immunology, Charité - University Medicine Berlin, Charitéplatz 1, Berlin 10117, Germany

Summary

CD4+ T lymphocytes are key to immunological memory. Here we show that in the memory phase of specific immune responses, most of the memory CD4+ T lymphocytes had relocated into the bone marrow (BM) within 3-8 weeks after their generation--a process involving integrin 2. Antigen-specific memory CD4+ T lymphocytes highly expressed Ly-6C, unlike most splenic CD44hiCD62L- CD4+ T lymphocytes. In adult mice, more than 80% of Ly-6ChiCD44hiCD62L memory CD4+ T lymphocytes were in the BM. In the BM, they associated to IL-7-expressing VCAM-1+ stroma cells. Gene expression and proliferation were downregulated, indicating a resting state. Upon challenge with antigen, they rapidly expressed cytokines and CD154 and efficiently induced the production of high-affinity antibodies by B lymphocytes. Thus, in the memory phase of immunity, memory helper Tcells are maintained in BM as resting but highly reactive cells in survival niches defined by IL-7-expressing stroma cells.

Tracking Naïve CD4 T Cells After They Leave The Thymus

A new paper in the Journal of Experimental Medicine reports that protein tyrosine kinase 7 (PTK7) is a surface marker that can be used to identify naïve CD4 T cells that have very recently exited the thymus (also called recent thymic emigrants or RTEs). Tracking thymic production of RTEs is important for monitoring immune reconstitution in many settings, including HIV infection, but reliable markers of this population have proven elusive. Christopher Haines and colleagues took the same type of approach that led to the identification of the molecule PD-1: they conducted gene expression studies of sorted CD4 T cell populations and found that PTK7 was significantly upregulated in thymocytes and neonatal CD4 T cells, but not adult CD4 T cells. Based on these findings, an antibody to PTK7 was used to quantify CD4 T cells expressing the protein on their surface. In an analysis of CD4 T cells sampled from 22 people across a wide range of ages (from 1 month to 61 years), PTK7-expressing naïve CD4 T cells were found to be abundant in neonates and levels subsequently declined progressively with aging.

The researchers also looked at the level of PTK7 expression on naïve T cells that either expressed or lacked the surface molecule CD31; loss of CD31 expression is associated with post-thymic proliferation of naïve CD4 T cells (see the free access review in the new issue of Blood, also appended below). Consistent with their other findings, PTK7 expression was restricted to CD31+ naïve CD4 T cells. Interestingly, the CD31+ naïve CD4 T cells could also be divided into two subsets based on whether they expressed PTK7, suggesting that CD31+ naïve T cells are not homogenous in terms of their proliferative history.

The study authors also report that PTK7+ naïve CD4 T cells are somewhat less efficient at mounting antigen-specific immune responses compared to PTK7- cells, due to delayed IL-2 production. They note that this may explain the poor Th1 CD4 T cell responses that have been described in neonates, because at this stage of development the vast majority of naïve CD4 T cells are RTEs expressing PTK7.

The Journal of Experimental Medicine

Published online January 26, 2009

doi:10.1084/jem.20080996

BRIEF DEFINITIVE REPORT

Human CD4+ T cell recent thymic emigrants are identified by protein tyrosine kinase 7 and have reduced immune function

Christopher J. Haines1, Thierry D. Giffon1, Li-Sheng Lu1, Xiaowei Lu2, Marc Tessier-Lavigne2, Douglas T. Ross3, and David B. Lewis1

1 Department of Pediatrics and the Immunology Program, 2 Department of Biological Sciences and Howard Hughes Medical Institute, and 3 Department of Biochemistry, Stanford University, Stanford, CA 94305

CD4+ recent thymic emigrants (RTEs) comprise a clinically and immunologically important T cell population that indicates thymic output and that is essential for maintaining a diverse β–T cell receptor (TCR) repertoire of the naive CD4+ T cell compartment. However, their frequency and function are poorly understood because no known surface markers distinguish them from older non-RTE naive CD4+ T cells. We demonstrate that protein tyrosine kinase 7 (PTK7) is a novel marker for human CD4+ RTEs. Consistent with their recent thymic origin, human PTK7+ RTEs contained higher levels of signal joint TCR gene excision circles and were more responsive to interleukin (IL)-7 compared with PTK7– naive CD4+ T cells, and rapidly decreased after complete thymectomy. Importantly, CD4+ RTEs proliferated less and produced less IL-2 and interferon- than PTK7– naive CD4+ T cells after β-TCR/CD3 and CD28 engagement. This immaturity in CD4+ RTE effector function may contribute to the reduced CD4+ T cell immunity observed in contexts in which CD4+ RTEs predominate, such as in the fetus and neonate or after immune reconstitution. The ability to identify viable CD4+ RTEs by PTK7 staining should be useful for monitoring thymic output in both healthy individuals and in patients with genetic or acquired CD4+ T cell immunodeficiencies.

Blood, 22 January 2009, Vol. 113, No. 4, pp. 769-774.

Prepublished online as a Blood First Edition Paper on June 26, 2008; DOI 10.1182/blood-2008-02-139154.

REVIEW ARTICLE

Life after the thymus: CD31+ and CD31– human naive CD4+ T-cell subsets

Siegfried Kohler1, and Andreas Thiel1,2

1 Clinical Immunology Group, Deutsches Rheuma-Forschungszentrum Berlin, Berlin; and 2 Regenerative Immunology and Aging, Berlin-Brandenburg Center for Regenerative Therapies, Berlin, Germany

Early in life, thymic export establishes the size and the diversity of the human naive T-cell pool. Yet, on puberty thymic activity drastically decreases. Because the overall size of the naive T-cell pool decreases only marginally during ageing, peripheral postthymic expansion of naive T cells has been postulated to account partly for the maintenance of T-cell immunity in adults. So far, the analysis of these processes had been hampered by the inability to distinguish recent thymic emigrants from proliferated, peripheral, naive T cells. However, recently, CD31 has been introduced as a marker to distinguish 2 subsets of naive CD4⁺ T cells with distinct T-cell receptor excision circle (TREC) content in the peripheral blood of healthy humans. Here, we review studies that have characterized TREC^hi CD31^{+ thymic}naive CD4⁺ T cells and have accordingly used the assessment of this distinct subset of naive CD4⁺ T cells as a correlate of thymic activity. We will discuss further potential clinical applications and how more research on CD31^{+ thymic}naive and CD31^{– central}naive CD4⁺ T cells may foster our knowledge of the impact of thymic involution on immune competence.

Cause for Caution on HIV Cure Report

An avalanche of media coverage has been loosed by the recently announced case of an individual who may have been “functionally cured” of HIV infection. The term functional cure has entered the lexicon due to the impossibility of formally proving that HIV has been entirely eradicated from the body; due to that limitation, long-term absence of detectable virus without therapy has been adopted as a reasonable definition of a cure, prefixed with the “functional” caveat.  The individual in this case is a 40 year old, HIV-infected American living in Berlin who had been on successful antiretroviral therapy prior to developing acute leukemia. The treatment for this condition involves bone marrow transplantation, which carries a 30% risk of mortality and is frequently associated with post-procedure complications. Due to the individual’s HIV infection, his doctors found a donor who was homozygous for the delta32 mutation, which completely abrogates expression of CCR5 (the major HIV co-receptor) on cells. Preparation for transplantation involves chemotherapy and radiation to essentially wipe out the immune system in order to prevent transplant rejection (with the salutary side effect of also depleting HIV-infected immune cells). The donor cells successfully engrafted but leukemia initially returned, requiring a second transplantation. Since that time – now close to two years ago – the individual has been free of leukemia, and HIV has remained undetectable without further antiretroviral treatment.

As can be gleaned from the press articles, opinions are divided on the significance of what has occurred. Some researchers have suggested it is a “proof of principle” that gene therapies with the capacity to block CCR5 expression could be curative. However, a 1999 review of bone marrow transplants in people with HIV (abstract below) identified two similar instances in which virus did not reappear after the procedure, so the contribution of the delta32 status of the donor in this current case is uncertain (although it is also possible that the prior examples also involved delta32 donors, unbeknownst to the doctors). The 1999 review also offers a grim perspective on the mortality associated with the procedure: the longest documented survival was around 300 days. While cynics might question whether AIDS professionals (including this writer) have their own self-serving reasons to express skepticism about cure claims, the complexity and danger of bone marrow transplantation clearly severely limits its use. It also must be stressed that while the individual is said to be “recovering,” there are no details available regarding his current health. 

Despite the many caveats, the case may be able to inform the pursuit of a safer curative strategies. The Foundation for AIDS Research (amfAR) has already sponsored a small meeting of experts to discuss the subject, attended by Mark Schoofs who wrote the first mainstream media article in the Wall Street Journal last week. One lesson may be that depleting HIV reservoirs to very low levels – if it can be done safely – will be beneficial, perhaps tipping the balance in favor of the host such that any residual virus can be controlled. If the individual remains well enough and is willing to undergo further evaluation, additional analyses to look for HIV in tissues will be important, along with evaluations of HIV-specific immunity. The presence of HIV-specific T cells carrying the donor delta32 mutation would suggest that sufficient viral activity has occurred after the transplantation procedure to induce new immune responses while, conversely, the absence of such responses might add to the evidence that HIV has been rendered completely inactive. Given that the case is now under the spotlight, the doctors involved will hopefully be forthcoming with updates as more information becomes available.

Med Hypotheses. 1999 Mar;52(3):247-57. Links

Could bone marrow transplantation cure AIDS?: review.

Huzicka I.

First Medical Faculty of Charles University, Prague, Czech Republic.

Despite recent use of potent antiretroviral drugs, the goal of eradication of human immunodeficiency virus (HIV) and restitution of the immune system has not been achieved. The present work reviews the literature on syngeneic or allogeneic bone marrow transplantation (BMT) in patients infected with HIV and in patients with acquired immune deficiency syndrome (AIDS). Thirty-two such attempts have been reported between 1982 and 1996. In two cases, HIV was eradicated and previously positive polymerase chain reaction (PCR) for viral DNA and RNA became negative. One patient had transient disappearance of HIV by PCR and several more, including one who underwent xenotransplantation of baboon bone marrow, experienced clinical and laboratory improvement. These results correlate with use of intensive pre-transplant cytoablative conditioning with chemotherapy and radiotherapy. Curative mechanisms of conditioning include elimination of several cell populations: infected cells, cells susceptible to infection, uninfected chronically activated cells responsible for autoimmune phenomena, and exhausted haemopoietic and lymphopoietic progenitors. They are repopulated by donor-derived cells that could mount a successful antiviral response through cytotoxic T lymphocytes. Non-specific graft-versus-host effect of allogeneic bone marrow would also help eliminate residual reservoirs of virus, especially macrophages. Protection of repopulating cells from infection could be achieved by a combination of antiretroviral agents and gene therapy strategies may be of additional benefit.

Barcode Surveillance Becomes a Reality – for T Cells

A variety of floridly paranoid conspiracy theories posit that plans are afoot to tag people with barcodes to track their movements. In the current issue of the Journal of Experimental Medicine, Koen Schepers and colleagues show that barcode surveillance can be employed for a more benign purpose. The study uses a technique called molecular barcoding to track the CD8 T cell response to infections, in order to investigate the extent to which the site of initial activation influences the trafficking of antigen-specific effector cells.

The researchers used a dual challenge model in which mice infected intranasally with influenza encoding one type of ovalbumin (OVA) antigen and a day later tumor cells encoding a different OVA antigen were injected subcutaneously. Two sets of CD8 T cells specific for each OVA antigen were transferred into the mice prior to challenge, each set tagged with molecular barcodes that could be identified using polymerase chain reaction (PCR) and microarray analyses. Five days after challenge, tagged CD8 T cells were largely localized to the lymph node draining the site of exposure (the lung-draining mediastinal lymph nodes and tumor-draining axillary/linguinal lymph nodes, respectively). But within 6-8 days post-challenge, the authors noted that a marked shift occurred and CD8 T cells containing different barcodes became intermingled in both sets of lymph nodes, indicating that proliferation had led to extensive systemic distribution of the responding cells.

To investigate whether the findings held true for the gut, an experiment was conducted using oral infection with listeria moncytogenes instead of influenza. In this case, there were mice that did show a biased accumulation of some listeria-specific CD8 T cells in the gut, suggesting that selective trafficking can occur under some circumstances. However, most of the antigen-specific CD8 T cells showed equal distribution at both sites.

Because these initial experiments involved a three day culture period to introduce the molecular barcodes, which might conceivably have affected trafficking properties, the researchers also developed a method to introduce the barcodes into thymocytes, to ensure that the resultant tagged CD8 T cells were naïve cells. Repeating the experiment using these naïve CD8 T cells produced very similar results. In discussing the results the authors state that: “collectively, these data indicate that after a brief period of local accumulation, the dominant pattern of accumulation at effector sites is aselective.” However, one question not answered by these data is whether the memory CD8 T cells generated by the challenge antigens show preferential migration to the site of initial activation when re-challenged; for researchers pondering whether vaccines need to induce memory responses capable of localizing at specific sites, this will be an important issue for future studies to address.

The Journal of Experimental Medicine, Vol. 205, No. 10, 2309-2318
Published online September 22, 2008
doi:10.1084/jem.20072462

ARTICLE

Dissecting T cell lineage relationships by cellular barcoding

Koen Schepers1, Erwin Swart1, Jeroen W.J. van Heijst1, Carmen Gerlach1, Maria Castrucci3, Daoud Sie2, Mike Heimerikx2, Arno Velds2, Ron M. Kerkhoven2, Ramon Arens1, and Ton N.M. Schumacher1

1 Division of Immunology and 2 Central Microarray Facility, the Netherlands Cancer Institute, 1066 CX Amsterdam, Netherlands
3 Istituto Superiore di Sanità, 00161 Roma, Italy

T cells, as well as other cell types, are composed of phenotypically and functionally distinct subsets. However, for many of these populations it is unclear whether they develop from common or separate progenitors. To address such issues, we developed a novel approach, termed cellular barcoding, that allows the dissection of lineage relationships. We demonstrate that the labeling of cells with unique identifiers coupled to a microarray-based detection system can be used to analyze family relationships between the progeny of such cells. To exemplify the potential of this technique, we studied migration patterns of families of antigen-specific CD8+ T cells in vivo. We demonstrate that progeny of individual T cells rapidly seed independent lymph nodes and that antigen-specific CD8+ T cells present at different effector sites are largely derived from a common pool of precursors. These data show how locally primed T cells disperse and provide a technology for kinship analysis with wider utility.

Second-Oldest HIV-1 Sequence Identified

A new study published online today in Nature reports the identification of the second-oldest known HIV-1 sequence. The sequence was recovered from a stored lymph tissue sample taken at a hospital in Kinshasa in 1960. The authors, led by Michael Worobey, were able to sequence around 5% of the HIV-1 genome and their analyses suggest that the virus began to spread and diversify in Africa around the turn of the century. The authors speculate that the growth of cities like Leopoldville facilitated an increase in transmission and the ultimate spread of HIV-1 across the globe. Due to the many potential pitfalls associated with attempting to recover viral sequences from ancient samples, the work was confirmed by an independent laboratory, and the long lag time between the submission of the paper to Nature and publication also suggests that a great deal of rigor went into assuring the paper's reviewers that the findings were accurate. In a news article for Science, Jon Cohen provides some additional background, noting that the tissue sample came from a 28-year old woman and was part of a group of samples taken from individuals with illnesses that had defied diagnosis, including lymph node abnormalities. Many more such samples are available for study, and Michael Worobey hopes to continue shedding light on HIV-1's early spread into the human population.

Nature 455, 661-664 (2 October 2008)
doi:10.1038/nature07390
Received 21 May 2008; Accepted 8 September 2008

Letter

Direct evidence of extensive diversity of HIV-1 in Kinshasa by 1960

Michael Worobey, Marlea Gemmel, Dirk E. Teuwen, Tamara Haselkorn, Kevin Kunstman, Michael Bunce, Jean-Jacques Muyembe, Jean-Marie M. Kabongo, Raphaël M. Kalengayi, Eric Van Marck, M. Thomas P. Gilbert & Steven M. Wolinsky

Human immunodeficiency virus type 1 (HIV-1) sequences that pre-date the recognition of AIDS are critical to defining the time of origin and the timescale of virus evolution1, 2. A viral sequence from 1959 (ZR59) is the oldest known HIV-1 infection1. Other historically documented sequences, important calibration points to convert evolutionary distance into time, are lacking, however; ZR59 is the only one sampled before 1976. Here we report the amplification and characterization of viral sequences from a Bouin's-fixed paraffin-embedded lymph node biopsy specimen obtained in 1960 from an adult female in Léopoldville, Belgian Congo (now Kinshasa, Democratic Republic of the Congo (DRC)), and we use them to conduct the first comparative evolutionary genetic study of early pre-AIDS epidemic HIV-1 group M viruses. Phylogenetic analyses position this viral sequence (DRC60) closest to the ancestral node of subtype A (excluding A2). Relaxed molecular clock analyses incorporating DRC60 and ZR59 date the most recent common ancestor of the M group to near the beginning of the twentieth century. The sizeable genetic distance between DRC60 and ZR59 directly demonstrates that diversification of HIV-1 in west-central Africa occurred long before the recognized AIDS pandemic. The recovery of viral gene sequences from decades-old paraffin-embedded tissues opens the door to a detailed palaeovirological investigation of the evolutionary history of HIV-1 that is not accessible by other methods.

Accompanying commentary:

Nature 455, 605-606 (2 October 2008)
doi:10.1038/455605a
Published online 1 October 2008

News and Views

AIDS: Prehistory of HIV-1

Paul M. Sharp & Beatrice H. Hahn

A Note of Caution on siRNA Studies

A new paper in the “Instant Online” section of Human Gene Therapy offers a cautionary tale about interpreting results of studies employing short interfering RNA (siRNA) as a therapeutic strategy. The authors report that these siRNAs typically trigger interferon production while the control siRNA used in many studies (at least 13, by their count) has unusually weak immune-stimulating properties. They also show that the anti-influenza activity reported for some siRNAs is entirely dependent on immune stimulation; modification of these constructs to abrogate their capacity to induce interferon caused a complete loss of the anti-influenza effect. The authors close by stressing that “to avoid the potential of misinterpreting therapeutic efficacy caused by siRNA-mediated immune stimulation for a specific RNAi effect in animal models, it is critical for researchers to appreciate the capacity for siRNA to activate such a response and to fully characterize the immunostimulatory potential of both active and control siRNA sequences.”

Free full text PDF: http://www.liebertonline.com/doi/pdfplus/10.1089/hgt.2008.131

Hum Gene Ther. 2008 Aug 19. [Epub ahead of print]

Misinterpreting the therapeutic effects of siRNA caused by immune stimulation.

Robbins M, Judge A, Ambegia E, Choi C, Yaworski E, Palmer L, McClintock K, Maclachlan I.
Protiva Biotherapeutics Inc., Burnaby, British Columbia, Canada.

Activation of innate immunity has direct effects in modulating viral replication, tumor growth, angiogenesis, inflammatory and other immunological processes. It is now established that unmodified siRNA can activate this innate immune response and therefore there is real potential for siRNA to elicit non-specific therapeutic effects in a wide range of disease models. Here we demonstrate that in a murine model of Influenza infection, the anti-viral activity of siRNA is primarily due to immune stimulation elicited by the active siRNA duplexes and not the result of therapeutic RNAi as previously reported. We show that the misinterpretation stems from the use of a particular control GFP siRNA that we identify as having unusually low immunostimulatory activity compared to the active anti-influenza siRNA. Curiously, this GFP siRNA has served as a negative control for a surprising number of groups reporting therapeutic effects of siRNA. The inert immunologic profile of the GFP sequence was unique among a broad panel of published siRNA, all of which could elicit significant Interferon induction from primary immune cells. This panel included 8 active siRNA against viral, angiogenic and oncologic targets whose reported therapeutic efficacy was based on comparison to the non-immunostimulatory GFP siRNA. These results emphasize the need for researchers to anticipate, monitor and adequately control for siRNA-mediated immune stimulation and calls into question the interpretation of numerous published reports of therapeutic RNAi in vivo. The use of chemically modified siRNA with minimal immunostimulatory capacity will help to more accurately delineate the mechanism of action underlying such studies.

CD4 T Cell Review

Jinfang Zhu and William Paul review the current state of knowledge regarding the differentiation of CD4 T cell subsets, in honor of the 50th Anniversary of the American Society for Hematology (ASH). Access to the full text is free. This issue of Blood also contains a complementary and similarly detailed review addressing the development and function of B cells by Tucker LeBien and Thomas Tedder.

Blood, 1 September 2008, Vol. 112, No. 5, pp. 1557-1569.

ASH 50TH ANNIVERSARY REVIEW

CD4 T cells: fates, functions, and faults

Jinfang Zhu1, and William E. Paul1

1 Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD

Abstract

In 1986, Mosmann and Coffman identified 2 subsets of activated CD4 T cells, Th1 and Th2 cells, which differed from each other in their pattern of cytokine production and their functions. Our understanding of the importance of the distinct differentiated forms of CD4 T cells and of the mechanisms through which they achieve their differentiated state has greatly expanded over the past 2 decades. Today at least 4 distinct CD4 T-cell subsets have been shown to exist, Th1, Th2, Th17, and iTreg cells. Here we summarize much of what is known about the 4 subsets, including the history of their discovery, their unique cytokine products and related functions, their distinctive expression of cell surface receptors and their characteristic transcription factors, the regulation of their fate determination, and the consequences of their abnormal activation.

Blood, 1 September 2008, Vol. 112, No. 5, pp. 1570-1580.

ASH 50TH ANNIVERSARY REVIEW

B lymphocytes: how they develop and function

Tucker W. LeBien1, and Thomas F. Tedder2

1 Department of Laboratory Medicine/Pathology, University of Minnesota Cancer Center, Minneapolis; and 2 Department of Immunology, Duke University Medical Center, Durham, NC

Abstract

The discovery that lymphocyte subpopulations participate in distinct components of the immune response focused attention onto the origins and function of lymphocytes more than 40 years ago. Studies in the 1960s and 1970s demonstrated that B and T lymphocytes were responsible primarily for the basic functions of antibody production and cell-mediated immune responses, respectively. The decades that followed have witnessed a continuum of unfolding complexities in B-cell development, subsets, and function that could not have been predicted. Some of the landmark discoveries that led to our current understanding of B lymphocytes as the source of protective innate and adaptive antibodies are highlighted in this essay. The phenotypic and functional diversity of B lymphocytes, their regulatory roles independent of antibody production, and the molecular events that make this lineage unique are also considered. Finally, perturbations in B-cell development that give rise to certain types of congenital immunodeficiency, leukemia/lymphoma, and autoimmune disease are discussed in the context of normal B-cell development and selection. Despite the significant advances that have been made at the cellular and molecular levels, there is much more to learn, and cross-disciplinary studies in hematology and immunology will continue to pave the way for new discoveries.

JACI Special Issue on AIDS, Prospects for Eradication Review

The Journal of Allergy & Clinical Immunology’s new issue has a special focus on HIV/AIDS, in recognition of the upcoming International AIDS Conference in Mexico City. There are several free full-text articles, including this review by Lin Shen and Robert Siliciano that summarizes recent evidence indicating ART can completely suppress viral replication, and mulls the implications for efforts to eradicate HIV infection.

Journal of Allergy & Clinical Immunology
Volume 122, Issue 1, Pages 22-28 (July 2008)

Viral reservoirs, residual viremia, and the potential of highly active antiretroviral therapy to eradicate HIV infection

Lin Shen, MD, Robert F. Siliciano, MD, PhD

Although highly active antiretroviral therapy (HAART) can reduce HIV-1 viremia to levels that are below the limit of detection of clinical assays, the virus persists in reservoirs, and trace levels of free virions can be found in the plasma. Whether this residual viremia represents ongoing cycles of replication continuing despite HAART or simply the release of virus from stable reservoirs has been controversial. Here we summarize the evidence that HAART can stop ongoing cycles of replication. The evidence comes from a detailed analysis of the residual viremia, which shows it to be archival and nonevolving in character. In addition, new pharmacodynamic measures incorporating a previously ignored slope parameter have provided the first real indication of how well HAART actually suppresses viral replication in vivo. Together, these results argue that the ultimate theoretical potential of HAART to control viral replication has already been reached. Progress toward eradication of the infection will require novel approaches to target the stable reservoirs that persist even when viral replication is completely halted.