CD4 T cells are a critical component of the immune system, coordinating and supporting the immune response to invading pathogens. It has been recognized for many years that the generation of effective antibodies by B cells depends on signals from CD4 T cells targeting the same pathogen, and over the past decade it has become clear that CD8 T cells – whose function is to recognize and kill pathogen-infected cells – also depend on support from CD4 T cells to perform their full spectrum of functions.
Importantly, the immune response to a pathogen develops in distinct stages: the first time you encounter a new pathogen (such as the measles virus which most of us are exposed to in childhood), “naïve” CD4 T cells, CD8 T cells and B cells (along with other components of the immune system) will respond. Naïve CD4 T cells, CD8 T cells and B cells are inexperienced or rookie cells that are on the lookout for pathogens that have not been encountered before. The naïve CD4 T cells, CD8 T cells and B cells that recognize measles virus become activated and undergo a burst of proliferation (copying themselves to make more measles-specific cells) which is typically accompanied by symptoms of acute infection such as fever & swollen lymph nodes. Some measles specific CD4 T cells communicate with B cells in a way that allows the production of increasingly effective measles-specific antibodies (this involves B cells switching from making immature IgM antibodies to more effective IgG antibodies) and other measles-specific CD4 T cells support the development of measles-specific CD8 T cells that can hunt down and eliminate any cells in the body infected with the virus.
Once the measles is controlled and eliminated, symptoms subside and most of the newly created measles-specific CD4 T cells, CD8 T cells and B cells die off in a process called “activation-induced cell death.” But a small population of each of these cell types survives and these cells are now described as “memory” cells. They can be thought of as the experienced veterans of the battle and they are retained by the body so that they can mount a rapid and effective response if the measles virus is ever encountered again. This transition from naïve to memory is a fundamental principle of immunology and it underlies the success of vaccines, which essentially trick naïve CD4 T cells, CD8 T cells and B cells (depending on the vaccine – not all of them can activate CD8 T cells) into responding to the vaccine like it’s the real thing, thereby generating a pool of memory CD4 T cells, CD8 T cells and B cells that can respond rapidly if the real pathogen is encountered later on (typically protecting against infection and disease).
The same principle applies to persistent infections like herpes zoster (the cause of chickenpox), CMV and Epstein-Barr virus which are not eliminated from the body but rather controlled by the memory CD4 T cells, CD8 T cells and B cells that develop when a person is first exposed.
One of the key questions in HIV research is: why don’t effective memory CD4 T cells, CD8 T cells and B cells develop and control HIV the way they so often do with other viruses? Two papers in the new issue of the Journal of Virology, both led by John Zaunders from the Centre for Immunology at St. Vincent's Hospital in Australia, take a detailed look at this question as it applies to CD4 T cells. One paper sets out to essentially answer the question: “what does the development of an effective, virus-specific memory CD4 T cell response look like in humans?” The researchers use immunization with vaccinia virus vaccine (Dryvax) as an in vivo model to facilitate the analysis of the developing CD4 T cell response from the earliest timepoints after vaccination through to the development of vaccinia-specific memory CD4 T cells.
The researchers report that between days 11 & 14 after immunization, there was a peak of activated, proliferating CD4 T cells that expressed high levels of the activation marker CD38 and the chemokine receptor CCR5. These cells represented the “primary” response, in other words they were naïve CD4 T cells specific for vaccinia that had become activated by the vaccine and undergone a burst of proliferation (such activated cells are also referred to as “effector” cells by immunologists). Although these CD4 T cells were proliferating in vivo, they were not capable of proliferating in vitro after restimulation with vaccinia. In terms of other functions, CD4 T cells that could produce the cytokine interferon-gamma appeared from day 11 onward, coinciding with the peak of activated proliferating CD4 T cells. About half of these interferon-gamma-producing CD4 T cells could also make the cytokine IL-2. Perhaps importantly, the researchers note that ~10-fold more CD4 T cells expressed activation markers than could make interferon-gamma in vitro. In other words, although it is almost certain that the activated CD4 T cells participating in the primary response were specific for vaccinia, this could not be formally demonstrated using standard assays measuring cytokine production.
The initial population of highly activated CD4 T cells disappeared fairly rapidly (there was a rise in the number of cells expressing markers of activation-induced cell death that peaked at day 14) and by day 14-21 the remaining vaccinia-specific CD4 T cells no longer expressed the activation marker CD38 and instead displayed many features consistent with “resting” (not activated) memory CD4 T cells. These features included the ability to proliferate in response to vaccinia in vitro and the secretion of a single cytokine, IL-2, compared to the preponderance of interferon-gamma production seen during the primary response. However, expression of the survival molecule Bcl-2 (typical of long-term memory CD4 T cells) was still relatively low at the last timepoint analyzed (day 21) suggesting that this duration of follow up might not have been sufficient to capture the full development (or “maturation” as it is sometimes called) of the vaccinia-specific memory CD4 T cell response. Another potential marker of long term memory is the receptor for the cytokine IL-7 (dubbed IL-7R or CD127); the authors note that a large proportion of the CD4 T cells that developed the ability to produce interferon gamma during the primary response expressed this receptor and it was retained on the memory CD4 T cells that developed subsequently.
By way of comparison, the second paper analyzed responding HIV-specific CD4 T cells in people with acute HIV infection. As this research group reported previously in the journal Blood, a population of HIV-specific CD4 T cells expressing high levels of CCR5 and CD38 emerged early and then declined rapidly 2-3 weeks after the onset of symptoms of acute infection. This CD4 T cell population displayed similar characteristics to the vaccinia-specific CD4 T cells that appeared during the primary response in the prior study. The researchers noted, however, that far fewer of these CD4 T cells expressed IL-7R and many more expressed an inhibitory receptor called CTLA-4 (this receptor is involved in curtailing the immune response) compared to the vaccinia-specific CD4 T cells.
The researchers stress that - perhaps as a consequence of these differences - a population of HIV-specific CD4 T cells displaying the features of long-term memory (such as IL-2 production and proliferation in response to in vitro stimulation) rarely develops in people with HIV, with the exception of unusual individuals who control their viral load very effectively. They suggest that the overexpression of CTLA-4 documented in this study may play a role in inhibiting the development of HIV-specific memory CD4 T cells. The relative paucity of IL-7R-expressing HIV-specific CD4 T cells may also be significant. The researchers note that both phenomena may relate to differences in the events that occur during the initial activation of naive HIV-specific CD4 T cells; this activation is driven by antigen-presenting dendritic cells and some studies have suggested that dendritic cell function is altered by HIV.
Another potentially important factor investigated in this study is the role of direct infection of developing HIV-specific CD4 T cells. To try and ascertain if the initial rapid decline of the highly activated CD4 T cell population was due to the normal process of activation-induced death or related to preferential infection with HIV, the investigators measured the number of copies of HIV DNA in the CD38-expressing CD4 T cell population. To their surprise (given that CD38 expression is strongly associated with expression of the HIV co-receptor CCR5), they found that the levels of infection were comparable in CD4 T cells expressing high and low levels of CD38. Based on data suggesting an average of 1.5 copies of HIV DNA per cell, the authors conclude that “there is probably an upper limit of about 10% infected cells in both the activated CD38+++ and non-activated CD38 dim CD4 T-cell subpopulations.”
The researchers also tried sorting CD4 T cells based on expression IL-7R, leading to another unanticipated finding: “Unexpectedly, purified CD127+ (IL-7R) CD4 T cells were found to contain a disproportionately higher number of copies of HIV-1 DNA, fivefold more on a per cell basis than the more-activated CD127-negative CD4 T cells.”
Taking these analyses together, it appears likely that the ~10% of CD38-expressing CD4 T cells that were infected with HIV represent the subset of these cells that also expressed IL-7R (previously identified as an early marker for developing long-term memory CD4 T cells). The implication, as the authors note in the discussion section of the paper, is that HIV may be preferentially infecting nascent HIV-specific memory CD4 T cells. The researchers plan to investigate this possibility in more detail in future studies. If HIV does preferentially infect developing HIV-specific memory CD4 T cells, this might offer another possible explanation for their failure to develop the full spectrum of functions typically associated with long-term memory CD4 T cell responses.
Another potentially interesting aspect of these data relates to the high levels of CD38 expression documented during the early T cell response. Although it is not discussed by the authors, CD38 expression is elevated throughout the course of progressive HIV infection and the levels of correlate with disease progression. As mentioned in the previous blog post, CD38 is the most commonly used marker for assessing levels of immune activation in people with HIV but it has never been clear which T cells are expressing the molecule, and why. The data in these studies suggest that the continuous activation of naïve T cells might contribute to the persistent presence of highly activated T cells expressing CD38 in the setting of HIV infection.
In conclusion, although these types of immunology studies can seem incredibly complex and arcane, attempting to delineate what goes right and what goes wrong during the immune response to HIV is a critical predicate for rationally designing effective vaccines and immunotherapies. They also have the potential to shed light on the still-obscure factors that drive HIV pathogenesis.
Abstracts:
Journal of Virology, October 2006, p. 10151-10161, Vol. 80, No. 20
CD127+CCR5+CD38+++ CD4+ Th1 Effector Cells Are an Early Component of the Primary Immune Response to Vaccinia Virus and Precede Development of Interleukin-2+ Memory CD4+ T Cells
John J. Zaunders, Wayne B. Dyer, Mee Ling Munier, Susanna Ip, Jie Liu, Elisabeth Amyes, William Rawlinson, Robert De Rose, Stephen J. Kent, John S. Sullivan, David A. Cooper, and Anthony D. Kelleher
The stages of development of human antigen-specific CD4+ T cells responding to viral infection and their differentiation into long-term memory cells are not well understood. The inoculation of healthy adults with vaccinia virus presents an opportunity to study these events intensively. Between days 11 and 14 postinoculation, there was a peak of proliferating CCR5+CD38+++ CD4+ effector cells which contained the cytotoxic granule marker T-cell intracellular antigen 1 and included gamma interferon (IFN-)-producing vaccinia virus-specific CD4+ T cells. The majority of these initial vaccinia virus-specific CD4+ T cells were CD127+ and produced interleukin-2 (IL-2) but not CTLA-4 in response to restimulation in vitro. Between days 14 and 21, there was a switch from IFN- and IL-2 coexpression to IL-2 production only, coinciding with a resting phenotype and an increased in vitro proliferation response. The early CCR5+CD38+++ vaccinia virus-specific CD4+ T cells were similar to our previous observations of human immunodeficiency virus (HIV)-specific CD4+ T cells in primary HIV type 1 (HIV-1) infection, but the vaccinia virus-specific cells expressed much more CD127 and IL-2 than we previously found in their HIV-specific counterparts. The current study provides important information on the differentiation of IL-2+ vaccinia virus-specific memory cells, allowing further study of antiviral effector CD4+ T cells in healthy adults and their dysfunction in HIV-1 infection.
Journal of Virology, October 2006, p. 10162-10172, Vol. 80, No. 20
Infection of CD127+ (Interleukin-7 Receptor+) CD4+ Cells and Overexpression of CTLA-4 Are Linked to Loss of Antigen-Specific CD4 T Cells during Primary Human Immunodeficiency Virus Type 1 Infection
John J. Zaunders, Susanna Ip, Mee Ling Munier, Daniel E. Kaufmann, Kazuo Suzuki, Choechoe Brereton, Sarah C. Sasson, Nabila Seddiki, Kersten Koelsch, Alan Landay, Pat Grey, Robert Finlayson, John Kaldor, Eric S. Rosenberg,3 Bruce D. Walker, Barbara Fazekas de St. Groth, David A. Cooper, Anthony D. Kelleher, on Behalf of the PHAEDRA Study Team,
We recently found that human immunodeficiency virus (HIV)-specific CD4+ T cells express coreceptor CCR5 and activation antigen CD38 during early primary HIV-1 infection (PHI) but then rapidly disappear from the circulation. This cell loss may be due to susceptibility to infection with HIV-1 but could also be due to inappropriate apoptosis, an expansion of T regulatory cells, trafficking out of the circulation, or dysfunction. We purified CD38+++CD4+ T cells from peripheral blood mononuclear cells, measured their level of HIV-1 DNA by PCR, and found that about 10% of this population was infected. However, a small subset of HIV-specific CD4+ T cells also expressed CD127, a marker of long-term memory cells. Purified CD127+CD4+ lymphocytes contained fivefold more copies of HIV-1 DNA per cell than did CD127-negative CD4+ cells, suggesting preferential infection of long-term memory cells. We observed no apoptosis of antigen-specific CD4+ T cells in vitro and only a small increase in CD45RO+CD25+CD127dimCD4+ T regulatory cells during PHI. However, 40% of CCR5+CD38+++ CD4+ T cells expressed gut-homing integrins, suggesting trafficking through gut-associated lymphoid tissue (GALT). Furthermore, 80% of HIV-specific CD4+ T cells expressed high levels of the negative regulator CTLA-4 in response to antigen stimulation in vitro, which was probably contributing to their inability to produce interleukin-2 and proliferate. Taken together, the loss of HIV-specific CD4+ T cells is associated with a combination of an infection of CCR5+ CD127+ memory CD4+ T cells, possibly in GALT, and a high expression of the inhibitory receptor CTLA-4.
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