Researchers Take a Measured Response

Immunologists compare data and techniques at Measurement of Antigen-Specific Immune Responses conference

By Adrian McDermott, PhD, and Philip Cohen, PhD

It makes good sense that a key component of the nascent Global HIV/AIDS Vaccine Enterprise scientific strategic plan is to expand the standardization of immunoassays. Some of the most fruitful collaborations so far within vaccine trial sponsor organizations, as well as between them, have been built on the widespread adoption of a standardized assay to measure HIV specific cellular immune responses in vaccinees.

Everyone agrees that such assay standardization is a requirement to speed progress toward an effective AIDS vaccine. But some immunologists question whether the current gold standard will suffice. It’s a debate that continues because the assay field is still trying to get a firm grasp on underlying immunological principles, and determine just what constitutes a good indicator of an effective immune response against HIV—that is, the elusive immune correlates of protection.

For that reason, assay development remains very much a research endeavor. The field is exploring improved technology to visualize different aspects of the immune system and developing new, more powerful techniques to explore the incredible diversity of cells engaged in the immune response and their range of functions. These goals are part of what drew HIV researchers to the first Measurement of Antigen-Specific Immune Responses (MASIR) conference which took place in January in Courmayeur, Italy. The meeting attracted over 30 speakers and 134 participants from all over the world, and from multiple disciplines and arenas of immunobiology.

Participants included scientists, clinicians and technologists from academia and industry with research interests running from cancer immunology to viral pathogenesis and disease. The goals of the attendees also were diverse. Some were eager to view immunity at higher resolution in order to develop simple, powerful tests to diagnose disease and guide treatment. Others see the new technological approaches as a way to extend their knowledge about the mechanism of immunity. The cross-disciplinary forum allowed disparate investigators to compare and contrast approaches and conclusions.

Much of the research presented at the meeting aimed to clarify the role of CD4+ and CD8+ T cells in different aspects of immune responses to different pathogens or diseases. These cells are stimulated when they encounter antigens in the form of proteins broken down into peptides. The peptide antigens are presented to the T cells after they are taken up by antigen presenting cells and folded into a protein complex on the cell surface known as the major histocompatibility complex (MHC). This MHC-peptide complex is recognized by the T-cell receptor (TCR), another protein complex on the surface of CD4+ and CD8+ T cells (Figure 1).


Figure 1. Probing the response of the immune system to HIV or SIV.HIV or SIV particles are taken up by antigen presenting cells (APCs) and their proteins processed into peptide antigens or epitopes. The peptides are transported to the APC surface bound in the major histocompatibility complex (MHC). The MHC-peptide complex is recognized by a T cell receptor (TCR). Immunologists use antibodes linked to fluorochromes to tag cells that produce certain factors (IFNγ and IL-2 antibodies shown) or identify cells that possess particular cell surface markers (not shown).

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Part of the challenge in deciphering immunity is the amazing complexity of T cells. Their surfaces can express a variety of markers that can be used to distinguish between different T cell populations such as CD8+ effector T cells, which react quickly to their target antigen, and CD8+ memory T cells, which are held in reserve, off-duty, in the thymus until the antigen they recognize makes a reappearance after an infection has been cleared. T cells are also capable of many different functions in response to antigen or other signals, ranging from the secretion of cytokines and chemokines that recruit other immune cells, to directly lysing infected cells. In addition, remarkable diversity comes from the TCRs, which are produced from genes assembled from genetic cassettes with the potential to recombine to form an estimated 25 million distinct molecules with different binding sites for MHC-peptide. To visualize some of that complexity, immunologists have relied on many standard molecular biology techniques such as monitoring gene regulation of T cells in response to different stages of infection and sequencing TCR genes. This work has benefited from advances in gene expression analysis (microarray) and sequencing which allow the rapid analysis of thousands of genes.

Another key technique is flow cytometry which can define the identity of cells, probe their function, and even separate cell populations for further analysis. In flow cytometry, cell surface proteins, or factors cells produce, are tagged with antibodies linked to different fluorescent color labels (Figure 1). Laser light is used to detect and quantify these signals or to sort cells into different populations for further analysis.

Only four of these fluorochromes have traditionally been available. But a hot topic at the meeting was the expansion of this tool kit, with 17 colors now available. Researchers have dubbed the new “multi-flavored” cytometry the “Baskin-Robbins” technique, after an American ice cream chain boasting 31 varieties. This advance is, of course, dependent on ingenious chemistry for new fluorochromes, but is also a testament to the plethora of markers that have been defined to characterize the T-cell type or function.

Some research presented at the MASIR meeting was geared toward finding novel proteins or genes that can be used as accurate indicators of the state of T cell activity. René van Lier from the Academic Medical Center in Amsterdam reported on his use of the cytomegalovirus (CMV) infection model to track down one such promising marker called IL-7Rα (CD127), a receptor which binds the cytokine interleukin (IL)-7. IL-7 is associated with maintenance and development of T-cell populations, especially naïve and memory subsets.

CMV, a herpesvirus, is ideally suited for studying virus-specific cell-mediated immune responses in healthy volunteers. The majority of adults over 40 years of age have been infected with CMV, regardless of socioeconomic or geographical location, and primary CMV infection is generally mild or asymptomatic. Normally, circulating virus cannot be detected and the virus remains latent, but CMV can reactivate if the host immune system is compromised by another virus infection such as HIV or through immunosuppressive drug therapy in preparation for a tissue transplant. When the virus is reactivated, CMV-specific CD8+ T cells mediate effective immunity.

To better understand why those responses are successful, van Lier used a combination of techniques to study the cellular genes recruited during the T-cell response in the maintenance of CMV latency. First he used flow cytometry to separate different subtypes of T cells, including effectors and memory cells. Next, he went on a genetic fishing expedition, using gene chip arrays representing all known human genes to compare which ones responded to a new CMV infection versus an infection transitioning to a dormant state. This complicated analysis revealed that production or expression of the IL-7Rα protein dropped in effector T cells during primary CMV infection when virus is actively replicating and virus load is high. In contrast, after the immune system had suppressed CMV viremia, the effector T cells expressed increased levels of IL-7Rα. Van Lier proposed there was an inverse relationship between IL-7Rα and virus load of CMV during the infection.

The utility of IL-7 receptors as an indicator of effector function doesn’t seem limited to CMV. In separate presentations using flow cytometry to explore cellular immune responses in HIV and tuberculosis patients, Brigitte Autran, Université Paris VI Pierre et Marie Curie, Paris, and Dirk Busch, Universität München, Munich, independently found that disappearance of IL-7 receptor from the T cell surface indicated active CD8+ T-cell effectors directed against the respective pathogens. Similar findings were recently reported by Rolf Zinkernagel and colleagues at the Institute of Experimental Immunology in Zurich, Switzerland (Eur. J. Immunol. 35, 738, 2005).

One reason the hunt for such new markers of immune function is heating up is the sense that the standard method for detection of immunological responses against cancer, bacterial or viral antigens doesn’t give the whole picture. Clinicians have relied on the detection of interferon (IFN) γ which is released by antigen-specific T cells when they recognize their target antigen. In most laboratories the IFNγ ELISPOT assay, which simply measures the number of T cells secreting IFNγ after binding to their cognate antigen peptides, is still the method of choice for screening immune responses, especially in developing countries. This technique is simple, quantifiable, robust, and relatively inexpensive.

However in her MASIR talk Sarah Rowland-Jones of Oxford University, UK, and MRC laboratories, Gambia, called into question the utility of IFNγ as a measure of the immune response in HIV infection. CD8+ T cells are thought to play a major role in control of HIV replication, but Rowland-Jones contended that neither the high numbers of circulating HIV-specific CD8+ T cells nor the magnitude of IFNγ responses necessarily correlated with viral load or clinical outcome.

Work presented by Guiseppe Pantaleo, CHUV, Lausanne, suggested that IFNγ measurements may be more useful if combined with detection of other markers of T cell function. His laboratory investigated the levels of IL-2 and IFNγ produced by both CD4+ and CD8+ T cell populations in conditions of acute (in Tetanus toxoid vaccinees), chronic (CMV, Epstein Barr virus [EBV], and herpes simplex virus [HSV]) or chronic persistent (HIV) immune responses. In general the production of IFNγ alone by antigen-specific T cells indicated the presence of high antigen load. Conversely, in conditions of low antigen load, an IL-2/IFNγ double secreting profile was observed. In acute CMV infection, for instance, he found a high frequency of CD8+ T cells only producing IFNγ. After one year of infection after the virus entered its latent phase the frequency of IL-2/IFNγ producing T cells greatly increased. Also, HIV-infected individuals whose virus is being suppressed with HAART were found to produce a dual functional IL-2/IFNγ response compared to those off therapy with higher viral counts. Pantaleo also presented data from a single patient who controlled virus replication for 3 years without HAART. The patient demonstrated the IL-2/IFNγ double response profile against the HIV-Gag protein compared with an IFNγ only response they displayed during primary infection. Pantaleo suggested that more multifunctional CD4+ and CD8+ T-cell responses are associated with control of virus replication and that IFNγ responses alone are associated with “lack of control.”

Richard Koup and Michael Betts from the Vaccine Research Center (VRC) in Maryland took up the theme of multi-functionality in their talk. They took advantage of the full power of 17-color flow cytometry to define different classes of T cells based on their surface proteins and investigate several functions of these T cells simultaneously in HIV-infected individuals. This included analysis of CD107a, a marker of cell lysis function by cytotoxic T lymphocytes, and production of factors that modulate immune function including IFNγ, TNFα IL-2 and MIP1β.

This study focused on a cohort of 79 HIV-infected patients. Some patients were termed progressors, meaning the virus had already succeeded in damaging their immune system so that they had a low level of circulating CD4+ T cells. Functional analysis of T cells from these individuals demonstrated a high frequency of CD107a, IFNγ and MIP1β expression but low IL-2 and TNFα. Another group of the patients were chosen because they possessed the human leukocyte antigen HLA-B57, which has been associated with maintenance of CD4+ T-cell counts and slow or non-progression to AIDS. In his analysis, Betts found that these individuals produced a high frequency of all the markers including IL-2 and TNFα.

Koup also used multicolored flow cytometry to analyze the functional immune responses elicited by immunization with a DNA vaccine composed of four plasmids (encoding envelopes from 3 different clades and a gag/pol/nefgene fusion) in comparison with those from HIV-nonprogressor individuals. Koup’s data showed that DNA immunization elicited CD4+ T cells that produced an IFNγ/IL-2 functional profile, whereas, the CD8+ T cells produced IFNγ alone. Koup noted that this particular DNA vaccine immunization elicited an intermediate T cell functional profile that lay between HIV-progressor and nonprogressor populations. His team will be using multi-parameter flow cytometry to analyze the immune response to different HIV immunization regimens so as to compare with the HIV nonprogressor functional profiles.

Krishna Komanduri of the MD Anderson Cancer Center says the VRC group’s and Pantaleo’s presentations demonstrate the wide range of approaches investigators are taking to characterize immune responses. “It was interesting on one end of the spectrum that some individuals are pursuing ‘extreme’ flow cytometry to characterize the extent of variation within functional T cells while others… have become reductionist, simplifying relevant T-cell function to IL-2/IFNγ secretion capability.”

Speakers at MASIR also considered the TCR characteristics that govern T cell interaction with the MHC-peptide complex. There is potential for a diversity of TCR rearrangements to recognize the same MHC peptide complex. TCRs consist of linked α and β proteins, encoded by genes which are assembled by the imprecise joining of the large number of variable (V), diversity (D, for β-chain only), and junctional (J) elements and the addition of extra nucleotides at the junctions that contributes to an enormous potential diversity. After a selection process which takes place in the thymus, mature T cells enter the periphery and form the preimmune repertoire available for recruitment in immune responses.

TCR repertoires in the periphery are selected by different antigens and have been found to vary widely in complexity. TCRs can also be grouped by the presence of specific motifs in the complimentarity-determining regions (CDRs) at the TCR VDJ regions at the β-loci (TCR Vβ), a region which contacts and recognizes peptide in the context of MHC.

Mark Wills of Cambridge University, UK used the CMV model to study the successful immune response exhibited by long term healthy CMV carriers. For example, Wills described two individuals who shared the HLA-B7 MHC allele and showed similar amino acid sequence motifs within different TCR Vβ families present. He noted that within three weeks CD8+ T-cell responses became highly focused and dominated by a few heavily expanded TCRs.

At a later presentation, David Price of the VRC examined the antigen-specific TCR repertoire at the very fine level of CDR structure that represent the contact points of the TCR with peptide. Sequencing analysis revealed that protection against CMV was conferred by T cell populations with diverse CDRs. Together, this pair of presentations suggested that getting a complete picture of the TCR repertoire warrants examination of both the αβ backbone and CDR. “No longer can we assume that a response to a given [peptide] epitope is an immunologic unit, but rather contains a diverse set of responses,” says Koup.

However, CMV is a DNA virus that demonstrates little sequence variation when compared to genetically unstable RNA retroviruses such as HIV or SIV. One of the significant stumbling blocks towards the development of an efficacious vaccine for HIV is that the virus can rapidly mutate leading to extensive sequence variation during an infection, which can disrupt recognition by T cells and result in lack of immune control.

In the non-human primate model, for example, single amino acid mutations within specific immunodominant peptide epitopes of the SIV Tat protein occur within as little as four weeks post infection and abrogate TCR recognition. But it’s intriguing that other immunodominant epitopes of the SIV Gag protein remain largely intact and thus remain targets for specific T-cell responses for a prolonged period. Investigators have attributed that difference to the relatively high ability of Tat to withstand structural change and yet maintain function.

Price and Daniel Douek of the VRC explored the role that the structure of the TCR plays in this process of viral escape. In rhesus macaque monkeys infected with a defined SIV isolate and possessing the same MHC background, the investigators sequenced 3,416 TCR sequences from CD8+ T-cell populations that recognize immunodominant peptides within the SIV Gag and Tat proteins.

It was known that the Gag-CM9 peptide mutates slowly after infection and exhibits escape late in infection. Price and Douek found that this relatively stable SIV peptide was recognized by a population of T cells similar to those that control CMV infection. They saw a reduced clonal diversity of TCR β-gene usage at 12 weeks and the Gag-CM9 specific T-cell CDRs were highly diverse, showing limited consensus motifs among the macaques analyzed. In contrast the Tat-TL8 peptide, which is known to exhibit rapid sequence variation consistent with escape shortly after infection, remained significantly more polyclonal at the β-loci after 12 weeks of infection. But examination of the precise molecular structural motifs within the T cell CDRs of the Tat-TL8-specific T cells revealed they were restricted in all 12 macaques studied.

Douek speculated that in contrast to the Gag-CM9 epitope, the change in the structure of the Tat-TL8 peptide would be significantly affected by amino acid variation, thereby altering the molecular structure of the peptide and subsequent recognition by the restricted CDR motif. It was concluded that diversity within the Gag-CM9 CDR motif confers CD8+ T-cell populations with promiscuous recognition properties such that they can tolerate a greater degree of sequence variation while maintaining effector function.

The MASIR conference highlighted one of the issues currently addressed by major HIV research groups, the ongoing hunt for relevant markers of effective immunity. While there is still much work to be done, one emerging lesson from all this research is that subtle intricacies of the immune response against HIV may be crucial and that the quality of an immune response is at least every bit as important as its magnitude. Mario Roederer of the VRC says MASIR represented an important opportunity to talk about these issues with a very broad range of researchers. “I think the highlight of the meeting was the interactions among people. It was incredibly friendly and [there was] much discussion after every talk and poster.”