TAG HIV Basic Science, Vaccines, and Cure Project Blog

The Impact of HIV on T Cell Priming

It has been known for decades that the development of a pathogen-specific immune response involves the activation (or “priming”) of naïve CD4 T cells. These cells subsequently divide extensively and, over a matter of weeks, generate memory CD4 T cells that coordinate ongoing surveillance against the pathogen (in many cases protecting against re-infection or, for some persistent infections such as CMV, keeping the pathogen in check for life). Based on this knowledge, it would be logical to surmise that many studies must have been conducted to investigate how this process plays out in HIV infection and the impact of the virus on HIV-specific memory CD4 T cell generation. Surprisingly, this is not the case. A new study in PLoS One represents the first published attempt to evaluate the effect of HIV on T cell priming in an in vitro model. This paper compliments the one detailed in vivo study conducted to date, which was published by Zohn Zaunders and colleagues in 2006.

The researchers, led by Rachel Lubon Sabado from NYU, established an in vitro system in which highly purified naïve CD4 T cells and antigen-presenting dendritic cells (both sampled from uninfected individuals) were exposed to live or inactivated HIV. The inactivated virus was created by chemical treatment with AT-2, a process developed by Larry Arthur and Jeff Lifson at NCI. After prolonged culture, both HIV-specific CD4 and, to a lesser extent, CD8 T cells could be generated. The magnitude of the response was lower when infectious virus was used, suggesting that viral replication impacts the memory T cell differentiation process. In terms of the HIV epitopes targeted, there was considerable overlap with the epitope-specific responses that have previously been described in vivo. The researchers identified some novel CD4 T cell responses against Env and Pol proteins, which led them to evaluate whether these responses might be seen in a cohort of acutely infected individuals. They found that almost all of the novel responses could be seen transiently during acute infection, but declined rapidly and became undetectable in chronic infection.

In discussing the findings, the study authors suggest that understanding why certain HIV-specific CD4 and CD8 T cell responses are rapidly lost in acute infection is an important goal for future research, particularly as long-term non-progressors tend to maintain responses better than individuals with progressive disease. They also note that AT-2-inactivated HIV may be a useful immunogen due to its ability to prime broad memory T cell responses from naïve cells.  

One other statement in the discussion is worth highlighting. The researchers write: “The continuous presence of viral antigens gives rise to new memory T cells from the existing naïve population as long as the immune system remains functionally intact and/or the existing naïve T cells can respond to HIV-antigens, thus explaining the highly heterogeneous population of HIV-specific T cells that exist in HIV-infected individuals.” Although this phenomenon has been alluded to in prior studies, the implications for HIV pathogenesis may deserve more careful consideration. In particular, ongoing activation of HIV-specific naïve T cells could contribute to the elevated levels of immune activation that are observed in HIV infection (these activated cells express very high levels of CD38). This would be consistent with the correlation between immune activation and naïve T cell depletion that has been reported in HIV infection. A recent basic immunology study also suggests that depletion of naïve T cells could contribute to microbial translocation, which in turn can further exacerbate immune activation and naïve T cell depletion by broadening the array of antigens being presented to include those from gut bacteria. 

PLoS ONE 4(1): e4256 doi:10.1371/journal.pone.0004256

In Vitro Priming Recapitulates In Vivo HIV-1 Specific T Cell Responses, Revealing Rapid Loss of Virus Reactive CD4+ T Cells in Acute HIV-1 Infection

Rachel Lubong Sabado1, Daniel G. Kavanagh2, Daniel E. Kaufmann2, Karlhans Fru4, Ethan Babcock1, Eric Rosenberg2, Bruce Walker2, Jeffrey Lifson3, Nina Bhardwaj1, Marie Larsson4*

1 New York University School of Medicine, New York, New York, United States of America, 2 Partners AIDS Research Center (PARC), Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, United States of America, 3 SAIC Fredrick, Inc., National Cancer Institute, Fredrick, Frederick, Maryland, United States of America, 4 Molecular Virology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden

Abstract

Background

The requirements for priming of HIV-specific T cell responses initially seen in infected individuals remain to be defined. Activation of T cell responses in lymph nodes requires cell-cell contact between T cells and DCs, which can give concurrent activation of T cells and HIV transmission.

Methodology

The study aim was to establish whether DCs pulsed with HIV-1 could prime HIV-specific T cell responses and to characterize these responses. Both infectious and aldrithiol-2 inactivated noninfectious HIV-1 were compared to establish efficiencies in priming and the type of responses elicited.

Findings

Our findings show that both infectious and inactivated HIV-1 pulsed DCs can prime HIV-specific responses from naïve T cells. Responses included several CD4+ and CD8+ T cell epitopes shown to be recognized in vivo by acutely and chronically infected individuals and some CD4+ T cell epitopes not identified previously. Follow up studies of acute and recent HIV infected samples revealed that these latter epitopes are among the earliest recognized in vivo, but the responses are lost rapidly, presumably through activation-induced general CD4+ T cell depletion which renders the newly activated HIV-specific CD4+ T cells prime targets for elimination.

Conclusion

Our studies highlight the ability of DCs to efficiently prime naïve T cells and induce a broad repertoire of HIV-specific responses and also provide valuable insights to the pathogenesis of HIV-1 infection in vivo.

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.

Mucosal Immune Responses in Elite Controllers

A paper recently published online by the journal Blood offers new data on HIV-specific CD8 T cell responses in the rectal mucosa of elite controllers (individuals who control HIV replication to undetectable levels in the absence of treatment). The senior investigator, Barbara Shacklett, generously took time out to discuss the study findings with TAG and the interview is posted on the our website.

Blood First Edition Paper, prepublished online December 23, 2008; DOI 10.1182/blood-2008-10-182709.

Mucosal immune responses to HIV-1 in elite controllers: A potential correlate of immune control

April L Ferre, Peter W. Hunt, J. William Critchfield, Delandy H Young, Megan M Morris, Juan C. Garcia, Richard B Pollard, Hal F Yee Jr., Jeffrey N Martin, Steven G Deeks, and Barbara L Shacklett

There exists a unique group of individuals who are able to durably control HIV in the absence of therapy. The mechanisms of control in these individuals remain poorly defined. In this study we examined CD8+ T-cell responses in blood and rectal mucosa from 17 "elite controllers" (viral load < 75 copies/ml), 11 "viremic controllers" (75-2,000 copies/mL), 14 non-controllers (>10,000 copies/mL), and 10 antiretroviral-treated individuals (<75 copies/mL). Production of IFN-, IL-2, TNF-, MIP-1, and CD107a by CD8+ T-cells in response to HIV-1 Gag stimulation was measured using flow cytometry. Our hypothesis was that 'polyfunctional' T-cells producing multiple antiviral factors would be most abundant in mucosal tissues of HIV controllers. Mucosal CD8+ T-cell responses were significantly stronger and more complex in controllers than in antiretroviral-suppressed individuals (P=0.0004). The frequency of 4-function responses in rectal mucosa was higher in controllers than in non-controllers and patients on therapy (P<0.0001). Mucosal responses in controllers were frequently stronger and more complex than blood responses. These findings demonstrate that many controllers mount strong, complex HIV-specific T-cell responses in rectal mucosa. These responses may play an important role in mucosal immune surveillance, as suggested by their relative enrichment among individuals who control HIV in the absence of therapy.

Accelerated Aging of the Immune System in HIV Infection

Over the years since AIDS was first recognized, a number of pathogenesis theories have posited that HIV infection causes an accelerated aging of the immune system. Some of the earliest, most hypothetical ideas along these lines suggested that the effects of HIV would be irreversible; thankfully, the success of antiretroviral therapies in restoring immune competence has since shown that this is not the case. Advances in the understanding of the immunological changes that accompany aging have recently allowed a more sophisticated and informed perspective on the parallels with HIV infection to be developed. At the forefront of this field of study is UCLA researcher Rita Effros, and the latest study from her group has just appeared online in the Journal of AIDS.

Fundamental to understanding this research is the division of the main cellular players of the human adaptive immune system (CD4 and CD8 T cells and B cells) into two large pools: naïve cells and memory cells. Naïve cells are those that have yet to encounter a pathogen or other antigen that they recognize, while memory cells are typically the descendants of naïve cells that have done battle with a pathogen (or a faux or attenuated pathogen in the form of a vaccine) in the past. Crucially, maintenance of naïve T cells depends on the output of newly produced cells from the thymus; production is robust in childhood but dwindles after adolescence and becomes a relative trickle in old age. Every infection and vaccination that recruits naïve T cells into becoming memory T cells over a lifetime drains the naïve pool, and while the thymus can “top up” the pool relatively quickly when young, this cannot be accomplished later in life and the ratio of naïve to memory cells gradually shifts as we age. In HIV infection, this process is accelerated, and naïve T cell (and B cell) numbers decline as disease progresses. In parallel, the proportion of memory cells showing evidence of exhaustion and dysfunction increases. Although the extent of the dysfunction is less dramatic, this accumulation of exhausted memory T cells is also seen in aging.

One key marker of T cell dysfunction in the elderly is the loss of the co-stimulatory molecule CD28. In their new paper, Effos and colleagues use data from the MACS cohort to show that faster disease progression in HIV infection is associated with more rapid accumulation of CD28-negative CD8 T cells, in tandem with more rapid loss of naïve CD8 T cells. Loss of CD28 expression on CD4 cells was also significantly associated with disease progression. Changes in naïve CD4 T cells were more subtle, as the relative proportion of naïve CD4 T cells to memory CD4 T cells in the peripheral blood did not decrease significantly as the absolute numbers of blood CD4 T cells declined. These findings are notably in line with the well-documented impact of age on the progression of HIV infection, because they suggest that older individuals are starting from a worse position immunologically (studies have consistently found that older individuals progress faster on average when compared to their younger counterparts).

In the discussion section of the paper, the authors state: “It has been suggested that the expansion of a few memory T-cell clones, directed at a restricted number of epitopes, fills up the ‘immunological space.’ Narrowing of the T-cell repertoire may subsequently impair the generation of new immune responses to infections and may also account for the close correlation between the high proportions of CD28-CD8+ T cells and reduced vaccine responses in the elderly. Therefore, despite a significant number of CD8 T cells observed during HIV-1 infection, the CD8+ T-cell compartment is mostly comprised of functionally defective CD28- T cells.”

The extent to which these immunological perturbations can be corrected by antiretroviral therapy is still not fully clear, but thymic output of naïve T cells has emerged as a clear correlate of a good immunological response to treatment. Conversely, slow naïve T cell repletion is associated with poor CD4 T cell count increases and a persistently elevated risk of clinical illness. One consequence of these findings is that therapies that might have the potential to boost naïve T cell production are being studied as potential immune-based therapies in HIV infection. Effros and colleagues also note that strategies to restore function to senescent T cells are under investigation, and approaches to deplete dysfunctional cells (with the aim of allowing functional cells to expand and take their place) are also being considered.

For further background on this topic, a webcast of a talk given by Rita Effros at last year's CROI is available online (under the Tuesday webcasts, it is the second presentation in the Symposium on Aging and AIDS). 

J Acquir Immune Defic Syndr. 2009 Jan 7. [Epub ahead of print]

Premature Aging of T cells Is Associated With Faster HIV-1 Disease Progression.

Cao W, Jamieson BD, Hultin LE, Hultin PM, Effros RB, Detels R.

OBJECTIVE: To determine if untreated HIV-1 infection and progression is associated with premature aging of memory CD8 and CD4 T cells and naive CD4 T cells. METHODS: Twenty HIV-1-infected fast progressors and 40 slow progressors were included in our study, using risk set sampling. The expression of cell surface markers reflecting the differentiation stages of lymphocytes was measured using flow cytometry analyses performed on cryopreserved peripheral blood mononuclear cells. RESULTS: We found that HIV-1 disease progression is associated with a decreased CD28 median florescence intensity on CD4 and CD8 T cells; an increased proportion of intermediate- and late-differentiated CD8 T cells and a decreased CD31 median florescence intensity on naive CD4 T cells of recent thymic origin. A selective depletion of peripherally expanded naive CD4 T cells was found to be associated with HIV-1 infection but not with HIV-1 disease progression. CONCLUSIONS: The overall change during HIV-1 infection and progression is associated with a shift in the T-cell population toward an aged conformation, which may be further compromised by impaired renewal of the less-differentiated CD4 T-cell population. Our results suggest that HIV-1 infection induces an accelerated aging of T lymphocytes, which is associated with the clinical progression to AIDS and death.

Merck PD-1 Paper Published

A new study of PD-1 blockade, conducted by researchers at the pharmaceutical company Merck, was published today in the Journal of Immunology. The paper describes Merck’s efforts to create antibodies with the ability to bind PD-1, which led to the selection of a mouse IgG antibody (named 1B8) for testing in the macaque model. Two macaque experiments were conducted: in the first, two groups of four animals with longstanding (>18 months) SIV infection received a single infusion of 1B8 at a dose of 5mg/kg. One group of animals were given the infusion 19 days prior to initiation of antiretroviral therapy (ART) with Merck’s integrase inhibitor, the second group were administered the antibody 12 days after starting ART. Macaques in the first group displayed a transient increase in viral load after antibody administration, similar to that described in the recent paper from researchers at Emory University. By day 19 when ART was started, viral loads had diverged: two of the macaques had levels above baseline (13.2- and 1.8-fold), two slightly below (0.4- and 0.2-fold, in one case below the 1,000 copy limit of the assay). After ART cessation at day 47, the one macaque with undetectable viral load maintained that level for at least nine days. By the last day of follow-up, however, viral loads in all animals had returned to pre-study baseline levels. One macaque that failed to fully control viral load on ART experienced disease progression at day 56 and subsequently had to be euthanized.

In the second group of macaques that received ART prior to PD-1 blockade, the impact of the integrase inhibitor alone on viral load was somewhat variable (at day 12 after ART initiation, viral load was increasing in one animal). After PD-1 blockade, two macaques showed transient increases in viral load after while the other two showed no change. One macaque maintained an undetectable viral load until near the end of ART administration on day 47. Similar to the group given blockade prior to ART, viral loads returned to pre-study baseline in all animals by day 124. The researchers evaluated the impact of PD-1 blockade on the magnitude and function of SIV-specific T cell responses, but no significant differences were observed. In terms of CD4 T cell counts, PD-1 blockade given prior to ART led to an increase in CD4 counts in 3/4 animals (before ART was started) but these increases were not sustained. In the group of animals receiving ART before blockade, CD4 counts increased but the effects of ART versus blockade cannot be distinguished.

The second macaque experiment described in the paper involved vaccination of uninfected animals with a suboptimal dose of an Ad5 vector encoding the SIV Gag protein. Results showed that PD-1 blockade administered at the time of the first immunization significantly increased the Gag-specific CD8 T cell response compared to controls that received the vaccine alone.

Overall, these results compliment but also differ from those obtained recently by researchers at Emory. The viral load reductions documented in the Emory paper were greater overall (2- to 10-fold) and were associated with significant increases in the magnitude and function of SIV-specific T cell and B cell responses, whereas increases in SIV-specific immune responses were not observed in this Merck study. One possible explanation for this difference is that the Emory study gave four infusions of PD-1 blockade over 10 days while the Merck experiments used just a single infusion. Other differences between the studies are that the anti-PD-1 antibody employed by Emory was partially humanized, while Merck employed a fully mouse antibody. The Merck researchers note that macaques in their study rapidly generated antibodies against the mouse antibody, as these antibodies (called rhesus anti-mouse IgG Abs or RAMAs) were detectable by ELISA within 10-14 days of blockade administration. The Emory researchers also saw antibodies arise targeting the mouse-derived parts of their PD-1 antibody. Both research groups stress the importance of developing humanized antibodies for future research; in the words of the Merck team: “One would expect enhanced blockade efficiency and immunological benefit through repeated administrations, ideally with a human-Fc chimeric or fully humanized mAb for optimal pharmacokinetics.”

The results obtained by Merck in the setting of vaccination also highlight the fact that – as prior studies have indicated - PD-1 plays a role in T cell activation as well as T cell exhaustion. As yet, this duality is poorly understood and needs elucidation. Although Merck’s development plans are not clear at this time, it is encouraging that a large pharmaceutical company is exploring the potential for T cell-reviving immune-based therapies. The paper’s authors, led by Adam Finnefrock, express hope that their work “will eventually lead to enhanced prophylactic and therapeutic vaccination modes as well as antiviral and antitumor therapies.”

The Journal of Immunology, 2009, 182: 980-987.

PD-1 Blockade in Rhesus Macaques: Impact on Chronic Infection and Prophylactic Vaccination

Adam C. Finnefrock1,*, Aimin Tang*, Fengsheng Li*, Daniel C. Freed*, Meizhen Feng, Kara S. Cox*, Kara J. Sykes*, James P. Guare, Michael D. Miller, David B. Olsen, Daria J. Hazuda, John W. Shiver, Danilo R. Casimiro* and Tong-Ming Fu1,*

* Vaccine Basic Research,   Antiviral Research, Medicinal Chemistry, and   Infectious Disease, Merck Research Laboratories, West Point, PA 19486

Programmed Cell Death 1 (PD-1) plays a crucial role in immunomodulation. Binding of PD-1 to its ligand receptors down-regulates immune responses, and published reports suggest that this immune modulation is exploited in cases of tumor progression or chronic viral infection to evade immune surveillance. Thus, blockade of this signal could restore or enhance host immune functions. To test this hypothesis, we generated a panel of mAbs specific to human PD-1 that block PD ligand 1 and tested them for in vitro binding, blocking, and functional T cell responses, and evaluated a lead candidate in two in vivo rhesus macaque (Macaca mulatta) models. In the first therapeutic model, chronically SIV-infected macaques were treated with a single infusion of anti-PD-1 mAb; viral loads increased transiently before returning to, or falling below, pretreatment baselines. In the second prophylactic model, naive macaques were immunized with an SIV-gag adenovirus vector vaccine. Induced PD-1 blockade caused a statistically significant (p < 0.05) increase in the peak percentage of T cells specific for the CM9 Gag epitope. These new results on PD-1 blockade in nonhuman primates point to a broader role for PD-1 immunomodulation and to potential applications in humans.