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Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
1 Correspondence: Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. E-mail: hrosenberg{at}niaid.nih.gov
Cytotoxic T lymphocyte antigen-4 (CTLA-4/CD152) is a member of the CD28 family of T cell effector antigens. Current belief, based primarily on results from gene-deleted mice, is that CTLA-4 promotes suppression of T cell proliferation. In this manuscript, Mark Slifka and colleagues compare antiviral responses of unmanipulated virus-specific CTLA-4+ and CTLA-4– T cells. The results of this work suggest that the CTLA-4 antigen has no specific role in suppressing T cell proliferation in response to viral stimulation.
Dr. Slifka, could you provide the readers of JLB a bit of background on CTLA-4, why it is believed to be an inhibitory molecule, and some of the basics of the essential controversy regarding the role of this molecule in T cell physiology?
MKS: CTLA-4 is a T cell surface antigen, a member of the Ig superfamily, and has been defined as a negative regulator, or inhibitor of T cell responses. In this role, it is perceived as counterbalancing CD28, a receptor that functions to promote T cell proliferation. Most of our understanding of CTLA-4 comes from the gene-deleted mice [1 2 3 ], which experience lethal lymphoproliferative disease; as such, the function of CTLA-4 has been defined as suppressing lymphocyte proliferation in vivo.
However, not all experimental results are consistent with this conclusion. Earliest studies actually suggested that CTLA-4 might serve as an activating antigen [4 ], and experiments with blocking antibodies or non-crosslinking Fab fragments have yielded conflicting results [reviewed in 5 ]. Taken even a step further, Waterhouse and colleagues have shown that CTLA-4-deficient T cells expressing transgenic TcR can be "cured" of lymphoproliferative disease [6 ]. Most recently, Homann and colleagues [7 ] used a mixed radiation and reconstitution approach and demonstrated no functional deficits among the CTLA-4 –/– cells. Yet many articles still state that CTLA-4 functions as a T cell suppressor molecule.
I came across CTLA-4 when I was looking for T cell markers that could distinguish between virus-specific T cell populations at different stages of maturation. As part of my post-doctoral work in Lindsay Whitton’s lab at Scripps, we were exploring the shift of virus-specific T cells from an activated to a memory state in response to acute LCMV infection [8 ]. What we found was that during days 5 and 6 after virus infection, points at which antiviral T cells are undergoing high rates of proliferation, 80%–90% express CTLA-4. In fact, we used CTLA-4 as an early activation marker, which would seem to be completely out of character for a molecule whose main role is to suppress proliferation.
This confounded my understanding of what CTLA-4 was supposed to be doing and there just seemed to be too many questions, exceptions, and caveats to the overall dogma regarding the absolute suppressor function of CTLA-4. In our group, we decided to take this question on using a largely unmanipulated host-pathogen model and let the T cells tell us whether or not CTLA-4 was suppressing them. In this work, we examined nontransgenic BALB/c and C57BL/6 mice to be sure that our results were not some type of strain-specific phenomenon. We used a natural mouse virus (LCMV) as well as vaccinia virus and also examined responses of both CD4+ and CD8+ T cells in order to reach our conclusions regarding the physiologic role of CTLA-4.
What do you think CTLA4’s true biologic role is?
MKS:At this point, it’s really hard to say. Right now, what we know seems to depend on the circumstances of the experiment and the perspective of the researcher performing the experiment. Much of what has been concluded in the literature regarding the role of CTLA-4 was based initially on the lymphoproliferative phenotype observed in CTLA-4 gene-deleted mice, although clearly other experiments have yielded different results. CTLA-4 does associate with a number of signaling molecules, including PI3K and Lck [9 ], so it may have a role in signal transduction. In the mainly unperturbed system used in these experiments, we can conclude that CTLA-4 is not serving to inhibit lymphocyte proliferation in the setting of natural mouse virus infection.
What sorts of reactions did you get when you first began to present your findings to colleagues?
MKS:Interestingly, I got what seemed to be highly polarized responses. Response #1: "Wow! I don’t believe it. This can’t be right." Response #2: "Of course CTLA-4 isn’t ALWAYS inhibitory, this isn’t anything new..." I think this indicates that the role of CTLA-4 is still a controversial subject and that the perspective on it seems to be evolving as new information is gathered.
Focusing more broadly on your work - you recently co-authored a manuscript in Viral Immunology with the title "Public fear of vaccination: separating fact from fiction [10 ]." This is a serious concern not only in the developing world, but for those of us in the "first world" as well. Could you review the main themes of this article for the readers of JLB?
MKS: Vaccination has an interesting psychology associated with it. In short, the better a given vaccine works, the less often people are inclined to value it. This is simply human nature. If there was an outbreak of measles or smallpox, there would be great outcry and clamor for these vaccines; yet, in the absence of a present and visible threat, interest clearly becomes limited. Added on to a lack of interest, there is the growing sense of doubt and in some cases outright paranoia and suspicion of vaccines—often propagated by misinformation on the Internet. As one example, there are a number of people who still believe that there is an association between the measles-mumps-rubella (MMR) vaccine and childhood autism despite well-powered, well-controlled studies that provide absolutely no support for this contention [11 ]. While anti-vaccine organizations have been around since Edward Jenner’s time (well before the existence of the Internet!), the best means to combat misinformation is education. We need to help the general public understand the value of vaccination, the concept of herd immunity, and to support the development and use of safe and effective vaccines.
I noticed that you also have a significant interest in smallpox. How does this fit into your research program?
MKS: One of the main themes in my laboratory program is the elucidation of mechanisms driving long-term immunity. As such, smallpox is a very interesting clinical story. Most people over 35 in the US have been vaccinated against smallpox, but most people under 35 have not, as mass vaccination efforts ceased once the World Health Organization officially declared the pathogen eradicated in 1980. Interestingly, a handful of articles suggested that immunity induced by the smallpox vaccine may only be protective for 3–5 years [12 , 13 ], despite published evidence, dating back to the early 1900s, suggesting that immunity is of long duration and might be lifelong [14 , 15 ]. I find it fascinating how much information can be gained from reading very early medical literature. For instance, Edward Jenner himself was a proponent of long-term immunological memory and demonstrated that protective immunity following smallpox vaccination (i.e., cowpox infection) could be maintained for up to 53 years. In terms of my entry into this field, I didn’t have any technical training in clinical research at the time, but I took a chance at moving into a completely new model and began studying long-term immunity against poxviruses in human subjects.
We really had an amazing time with our first study on smallpox vaccination. We began working on this project shortly after 9/11 and the ensuing anthrax scare, and there was a sense of community and willingness to help that I will never forget. At that time, we didn’t have direct funding for the project and so I used part of my university start-up funds to pay for the experiments, and people here in Oregon and Washington lined up to give blood samples without financial compensation—making the project feasible since we were operating on a shoe-string budget. At one point, a local news station covered our story and by that evening, we had literally hundreds of phone calls from people who wanted to join the study and do their part to determine if immunity against smallpox could be maintained long-term or if we were completely vulnerable to a smallpox attack. State and county health care professionals also pitched in and volunteered blood samples so in the end, we were able to evaluate more than 300 individuals who had experienced 1, 2, 3–5, or 6–14 vaccinations, ranging in time from 30 days to 75 years (as well as 26 controls who had never been vaccinated). Among our findings, we determined that antiviral immunity could be maintained for up to 75 years with antibody responses remaining stable over time and T cell responses declining slowly, with a half-life of 8–15 years [15 , 16 ].
As a relatively young scientist, do you see the hurdles that you face as different from those met by more senior scientists? What would you define as most enjoyable aspects of your work?
MKS: I think that young investigators are facing some really tough challenges—especially in the amount of time and effort it takes to get initial funding. I read recently that the average researcher is now 42 years old at the time that he/she receives a first RO1. This is approaching the midpoint of a scientific career and seems to be contributing to the "graying of the NIH" as some people describe it. I like some of the new approaches that the NIH is taking to help young investigators, such as alternate paylines and new funding opportunities that specifically target young investigators and I hope that these strategies begin to work soon—I have spoken to more than a few postdocs who are concerned with the academic path because of the current funding environment.
In terms of what I most enjoy about the work that we are doing, I would say that it is the challenge of finding meaningful patterns in host-pathogen interactions and determining specific mechanisms that form the basis of long-term immunity. I feel very fortunate to have the chance to compare and contrast experimental results from both animal models as well as clinical studies because there is much to be learned from both sides of the fence.
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