TAG HIV Basic Science, Vaccines, and Cure Project Blog

Attack of the Killer Helpers (Part Two)

Among the many tasks of the immune system, the responsibility for recognizing and killing virus-infected cells falls largely to the subset of CD8 T cells designated cytotoxic T-lymphocytes (CTL). Whether CD4 T cells (traditionally called just “helper” cells) can exert cytotoxic functions has historically been controversial, but over the past decade studies have convincingly documented the existence of cytotoxic CD4 T-cell responses in a variety of different settings, including HIV and SIV infection (as previously covered on this blog). A new paper in the open-access journal Retrovirology from Jonah Sacha’s research group at Oregon Health and Science University now reports that not only are cytotoxic CD4 T-cell responses detectable in macaques controlling a pathogenic SIV isolate, but they can drive the selection of immune escape mutations. As the authors note, this represents compelling evidence that CD4 T cells can directly suppress viral replication.

Although I neglected to highlight it on the blog at the time of publication, a human study from the laboratory of Hendrick Streeck also argues for a key role of cytotoxic CD4 T-cell responses in controlling HIV. Published in Science Translational Medicine back in February of this year, the study showed that HIV-specific cytotoxic CD4 T-cell activity predicted superior control of viral load and slower disease progression (as assessed by time to CD4 T-cell count <350, time to ART initiation, or time to viral load >100,000 copies/mm³) in a cohort of acutely HIV-infected individuals.

Taken together, these results further underscore the importance of considering virus-specific CD4 T-cell responses, both in studies of natural control of HIV replication and in attempts to design effective immune-based therapies.

Retrovirology. 2012 Nov 6;9:91. doi: 10.1186/1742-4690-9-91.

CD8+ and CD4+ cytotoxic T cell escape mutations precede breakthrough SIVmac239 viremia in an elite controller.

Burwitz BJ, Giraldo-Vela JP, Reed J, Newman LP, Bean AT, Nimityongskul FA, Castrovinci PA, Maness NJ, Leon EJ, Rudersdorf R, Sacha JB.

Vaccine and Gene Therapy Institute, Oregon Health and Science University, 505 NW 185th, Beaverton, OR, 97006, USA.

Abstract

BACKGROUND: Virus-specific T cells are critical components in the containment of immunodeficiency virus infections. While the protective role of CD8+ T cells is well established by studies of CD8+ T cell-mediated viral escape, it remains unknown if CD4+ T cells can also impose sufficient selective pressure on replicating virus to drive the emergence of high-frequency escape variants. Identifying a high frequency CD4+ T cell driven escape mutation would provide compelling evidence of direct immunological pressure mediated by these cells. RESULTS: Here, we studied a SIVmac239-infected elite controller rhesus macaque with a 1,000-fold spontaneous increase in plasma viral load that preceded disease progression and death from AIDS-related complications. We sequenced the viral genome pre- and post-breakthrough and demonstrate that CD8+ T cells drove the majority of the amino acid substitutions outside of Env. However, within a region of Gag p27CA targeted only by CD4+ T cells, we identified a unique post-breakthrough mutation, Gag D205E, which abrogated CD4+ T cell recognition. Further, we demonstrate that the Gag p27CA-specific CD4+ T cells exhibited cytolytic activity and that SIV bearing the Gag D205E mutation escapes this CD4+ T cell effector function ex vivo. CONCLUSIONS: Cumulatively, these results confirm the importance of virus specific CD8+ T cells and demonstrate that CD4+ T cells can also exert significant selective pressure on immunodeficiency viruses in vivo during low-level viral replication. These results also suggest that further studies of CD4+ T cell escape should focus on cases of elite control with spontaneous viral breakthrough.

Sci Transl Med. 2012 Feb 29;4(123):123ra25. doi: 10.1126/scitranslmed.3003165.

HIV-specific cytolytic CD4 T cell responses during acute HIV infection predict disease outcome.

Soghoian DZ, Jessen H, Flanders M, Sierra-Davidson K, Cutler S, Pertel T, Ranasinghe S, Lindqvist M, Davis I, Lane K, Rychert J, Rosenberg ES, Piechocka-Trocha A, Brass AL, Brenchley JM, Walker BD, Streeck H.

Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard Medical School, Charlestown, MA 02129, USA.

Abstract

Early immunological events during acute HIV infection are thought to fundamentally influence long-term disease outcome. Whereas the contribution of HIV-specific CD8 T cell responses to early viral control is well established, the role of HIV-specific CD4 T cell responses in the control of viral replication after acute infection is unknown. A growing body of evidence suggests that CD4 T cells-besides their helper function-have the capacity to directly recognize and kill virally infected cells. In a longitudinal study of a cohort of individuals acutely infected with HIV, we observed that subjects able to spontaneously control HIV replication in the absence of antiretroviral therapy showed a significant expansion of HIV-specific CD4 T cell responses-but not CD8 T cell responses-compared to subjects who progressed to a high viral set point (P = 0.038). Markedly, this expansion occurred before differences in viral load or CD4 T cell count and was characterized by robust cytolytic activity and expression of a distinct profile of perforin and granzymes at the earliest time point. Kaplan-Meier analysis revealed that the emergence of granzyme A(+) HIV-specific CD4 T cell responses at baseline was highly predictive of slower disease progression and clinical outcome (average days to CD4 T cell count <350/μl was 575 versus 306, P = 0.001). These data demonstrate that HIV-specific CD4 T cell responses can be used during the earliest phase of HIV infection as an immunological predictor of subsequent viral set point and disease outcome. Moreover, these data suggest that expansion of granzyme A(+) HIV-specific cytolytic CD4 T cell responses early during acute HIV infection contributes substantially to the control of viral replication.

T Follicular Helper Cells and HIV

T follicular helper cells (TFH) are a subset of CD4 T cells that reside almost exclusively in the lymph nodes, where they specialize in helping B cells generate antibodies. In a testament to the complexity and difficulty of studying the immune system, TFH were only identified as a discrete subset of CD4 T cells at the turn of the millennium. Since that time, several features that define TFH have been described, including high levels of the chemokine receptor CXCR5, expression of the PD-1 molecule, secretion of the cytokine IL-21, and upregulation of the Bcl-6 gene. Studies have also found TFH to be important for driving the process of affinity maturation (wherein B cells producing antibodies with increased affinity for their antigen target are progressively generated and selected), and for supporting the development of long-lived memory B cells.

Because TFH are relatively new on the scene, very little is known about how they are affected by HIV infection. A few months ago in the Journal of Clinical Investigation, two research groups published studies shedding light on this question. Madelene Lindqvist and colleagues looked at TFH in people with HIV and found they were significantly expanded in lymph nodes, with a major contribution from HIV-specific TFH (predominantly targeting the Gag protein). TFH levels were approximately tenfold higher in HIV-positive individuals compared to HIV-negative controls. Although high viral load was associated with around double the levels of TFH compared to undetectable viremia on ART, there was no direct correlation between viral load levels and TFH numbers. Notably, the extent of TFH expansion correlated with hypersecretion of antibodies by B cells, strongly suggesting that dysregulation of TFH contributes to B-cell dysfunction and hypergammaglobulinemia in people with HIV; these features of the infection were first reported in the early 1980s, but the underlying mechanisms have been unclear. Constantinos Petrovas and colleagues report broadly similar findings from the SIV/macaque model, additionally providing evidence that TFH are susceptible to infection.

In an accompanying commentary, TFH expert Carola Vinuesa highlights a number of important questions pertaining to this CD4 T-cell subset in HIV infection. Firstly, it has become apparent that several of the rare antibodies capable of broad neutralizing activity against multiple HIV isolates have undergone an unusual degree of affinity maturation, suggesting that interactions between TFH and B cells were essential in supporting their development; a better understanding of how this occurs could prove essential to designing an efficacious vaccine. The other side of this equation is that most individuals with HIV fail to generate effective neutralizing antibodies, and it will also be important to learn how TFH dysregulation contributes to this failure. From the standpoint of cure research, Vinuesa notes that these studies imply that TFH could contribute significantly to the persistent reservoir of HIV-infected cells and may therefore require specific targeting by reservoir-eliminating strategies.

Several webcasts of scientific talks relating to TFH and HIV are available free online. At CROI in March 2012, Carola Vinuesa provided an introductory overview and Madelene Lindqvist and Constantinos Petrovas presented some of the results described in these papers. In September at AIDS Vaccine 2012, Hendrick Streeck discussed the latest findings from his laboratory and Rick Koup offered a perspective on TFH and vaccines.

CROI 2012 

Symposium: Ontogeny of a Protective Immune Response to HIV (3rd presentation in session)

Follicular Helper and Regulatory T Cells Control the Quality of Antibody Responses

Carola Vinuesa, John Curtin Sch of Med Res, Australian Natl Univ, Canberra, Australia

Oral Abstract: Immune, Cellular, and Viral Factors Influencing HIV–Host Interplay (5th and 6th presentations in session) 

SIV Infection Affects the Function, but not Survival, of Follicular T Helper Cells in Rhesus Macaques

Constantinos Petrovas, Vaccine Res Ctr, NIAID, NIH, Bethesda, MD, US

HIV-specific T Follicular Helper Cell Responses in Chronically Infected Individuals

Madelene Lindqvist, Ragon Inst of MGH, MIT, and Harvard, Boston, MA, US

AIDS Vaccine 2012

T Follicular Helper Cells in HIV Infection

Hendrik Streeck, Ragon Institute of MGH, MIT, and Harvard, Boston, MA, US

Follicular T helper cells in vaccination and lentivirus pathogenesis

Richard A. Koup, Vaccine Research Center, Bethesda, MD, US

 

Journal of Clinical Investigation abstracts: 

Published in Volume 122, Issue 9 (September 4, 2012)

J Clin Invest. 2012;122(9):3271–3280. doi:10.1172/JCI64314.

Research Article

Expansion of HIV-specific T follicular helper cells in chronic HIV infection

Madelene Lindqvist1, Jan van Lunzen2, Damien Z. Soghoian1, Bjorn D. Kuhl1, Srinika Ranasinghe1, Gregory Kranias1, Michael D. Flanders1, Samuel Cutler1, Naomi Yudanin3, Matthias I. Muller1, Isaiah Davis1, Donna Farber3, Philip Hartjen2, Friedrich Haag4, Galit Alter1, Julian Schulze zur Wiesch2 and Hendrik Streeck1

1Ragon Institute of MGH, MIT, and Harvard Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

2Infectious Diseases Unit, University Medical Center Eppendorf, Hamburg, Germany.

3Columbia Center for Translational Immunology, Columbia University, New York, New York, USA.

4Department of Immunology, University Medical Center Eppendorf, Hamburg, Germany. 

Authorship note: Madelene Lindqvist, Jan van Lunzen, and Hendrik Streeck contributed equally to this work.

HIV targets CD4 T cells, which are required for the induction of high-affinity antibody responses and the formation of long-lived B cell memory. The depletion of antigen-specific CD4 T cells during HIV infection is therefore believed to impede the development of protective B cell immunity. Although several different HIV-related B cell dysfunctions have been described, the role of CD4 T follicular helper (TFH) cells in HIV infection remains unknown. Here, we assessed HIV-specific TFH responses in the lymph nodes of treatment-naive and antiretroviral-treated HIV-infected individuals. Strikingly, both the bulk TFH and HIV-specific TFH cell populations were significantly expanded in chronic HIV infection and were highly associated with viremia. In particular, GAG-specific TFH cells were detected at significantly higher levels in the lymph nodes compared with those of GP120-specific TFH cells and showed preferential secretion of the helper cytokine IL-21. In addition, TFH cell expansion was associated with an increase of germinal center B cells and plasma cells as well as IgG1 hypersecretion. Thus, our study suggests that high levels of HIV viremia drive the expansion of TFH cells, which in turn leads to perturbations of B cell differentiation, resulting in dysregulated antibody production.

Published in Volume 122, Issue 9 (September 4, 2012)

J Clin Invest. 2012;122(9):3281–3294. doi:10.1172/JCI63039.

Research Article

CD4 T follicular helper cell dynamics during SIV infection

Constantinos Petrovas1, Takuya Yamamoto1, Michael Y. Gerner2, Kristin L. Boswell1, Kaska Wloka1, Emily C. Smith1, David R. Ambrozak1, Netanya G. Sandler3, Katherina J. Timmer3, Xiaoyong Sun4, Li Pan4, Amanda Poholek2, Srinivas S. Rao5, Jason M. Brenchley6, S. Munir Alam7, Georgia D. Tomaras7, Mario Roederer8, Daniel C. Douek3, Robert A. Seder9, Ronald N. Germain2, Elias K. Haddad4 and Richard A. Koup1

1Immunology Laboratory, Vaccine Research Center,

2Laboratory of Systems Biology, Lymphocyte Biology Section, and

3Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), NIH, Bethesda, Maryland, USA.

4VGTI Florida, Port St. Lucie, Florida, USA.

5Laboratory Animal Medicine, Vaccine Research Center, and

6Laboratory of Molecular Microbiology, NIAID, NIH, Bethesda, Maryland, USA.

7Duke Human Vaccine Institute, Duke University Medical School, Durham, North Carolina, USA.

8ImmunoTechnology Section and

9Cellular Immunology Section, Vaccine Research Center, NIAID, NIH, Bethesda, Maryland, USA.

CD4 T follicular helper (TFH) cells interact with and stimulate the generation of antigen-specific B cells. TFH cell interaction with B cells correlates with production of SIV-specific immunoglobulins. However, the fate of TFH cells and their participation in SIV-induced antibody production is not well understood. We investigated the phenotype, function, location, and molecular signature of TFH cells in rhesus macaques. Similar to their human counterparts, TFH cells in rhesus macaques represented a heterogeneous population with respect to cytokine function. In a highly differentiated subpopulation of TFH cells, characterized by CD150lo expression, production of Th1 cytokines was compromised while IL-4 production was augmented, and cells exhibited decreased survival, cycling, and trafficking capacity. TFH cells exhibited a distinct gene profile that was markedly altered by SIV infection. TFH cells were infected by SIV; yet, in some animals, these cells actually accumulated during chronic SIV infection. Generalized immune activation and increased IL-6 production helped drive TFH differentiation during SIV infection. Accumulation of TFH cells was associated with increased frequency of activated germinal center B cells and SIV-specific antibodies. Therefore, chronic SIV does not disturb the ability of TFH cells to help B cell maturation and production of SIV-specific immunoglobulins.

Published in Volume 122, Issue 9 (September 4, 2012)

J Clin Invest. 2012;122(9):3059–3062. doi:10.1172/JCI65175.

Commentary

HIV and T follicular helper cells: a dangerous relationship

Carola G. Vinuesa

Department of Pathogens and Immunity, John Curtin School of Medical Research, Australian National University, Canberra, Australia.

HIV infection leads to progressive destruction of infected CD4 T cells, hypergammaglobulinemia, and loss of memory B cells. Germinal centers, which are key to memory B cell formation and protective antibody responses, are major HIV reservoirs in which the virus replicates within T follicular helper (TFH) cells. In this issue of the JCI, the Koup and Streeck groups report that chronic SIV/HIV infection promotes TFH cell accumulation, which may drive B cell dysregulation. Their discoveries suggest that HIV harnesses TFH cells to evade the antibody response.

Dodging HIV Variability by Targeting HERVs

In prior posts I’ve written about research from the laboratories of Mario Ostrowski and Doug Nixon looking at the effect of HIV infection on human endogenous retrovirus (HERV) protein expression. This work, led by Brad Jones and Keith Garrison, has shown that the liberating effects of the HIV Vif protein can cause normally inactive HERV genetic sequences to produce proteins in cells infected by HIV. These proteins can then be processed and presented as antigens and, as a consequence, HERV-specific CD8 T-cell responses are detectable in some individuals with HIV. However, up until now there has been no direct evidence that HERV-specific CD8 T-cell responses can actually recognize and kill HIV-infected CD4 T cells. A paper just published in the Journal of Clinical Investigation addresses this information gap, demonstrating that CD8 T cells specific for a HERV-K(HML-2) epitope are able to recognize and kill CD4 T cells infected with an array of HIV-1, HIV-2, and SIV isolates (albeit with varying levels of effectiveness depending on the specific isolate).

Based on their findings, the researchers suggest that induction of HERV-K(HML-2)-specific CD8 T-cell responses using vaccines may offer a way to target HIV-infected cells without having to deal with the problem of HIV’s genetic variability. One serious concern with this approach is that, if HERV-K(HML-2) proteins are ever expressed by healthy cells, autoimmunity could ensue. But so far, the authors note, “expression of HERV-K(HML-2)-Gag and -Env protein has not been convincingly demonstrated in any healthy adult human tissue, despite extensive screening.” Additional studies are now planned to assess whether targeting HERV-K(HML-2) might have potential in the context of both therapeutic and preventive HIV vaccine strategies.  

J Clin Invest. 2012 Nov 12. pii: 64560. doi: 10.1172/JCI64560. [Epub ahead of print]

HERV-K-specific T cells eliminate diverse HIV-1/2 and SIV primary isolates.

Jones RB, Garrison KE, Mujib S, Mihajlovic V, Aidarus N, Hunter DV, Martin E, John VM, Zhan W, Faruk NF, Gyenes G, Sheppard NC, Priumboom-Brees IM, Goodwin DA, Chen L, Rieger M, Muscat-King S, Loudon PT, Stanley C, Holditch SJ, Wong JC, Clayton K,Duan E, Song H, Xu Y, Sengupta D, Tandon R, Sacha JB, Brockman MA, Benko E, Kovacs C, Nixon DF, Ostrowski MA.

Abstract

The genetic diversity of HIV-1 represents a major challenge in vaccine development. In this study, we establish a rationale for eliminating HIV-1-infected cells by targeting cellular immune responses against stable human endogenous retroviral (HERV) antigens. HERV DNA sequences in the human genome represent the remnants of ancient infectious retroviruses. We show that the infection of CD4+ T cells with HIV-1 resulted in transcription of the HML-2 lineage of HERV type K [HERV-K(HML-2)] and the expression of Gag and Env proteins. HERV-K(HML-2)-specific CD8+ T cells obtained from HIV-1-infected human subjects responded to HIV-1-infected cells in a Vif-dependent manner in vitro. Consistent with the proposed mode of action, a HERV-K(HML-2)-specific CD8+ T cell clone exhibited comprehensive elimination of cells infected with a panel of globally diverse HIV-1, HIV-2, and SIV isolates in vitro. We identified a second T cell response that exhibited cross-reactivity between homologous HIV-1-Pol and HERV-K(HML-2)-Pol determinants, raising the possibility that homology between HIV-1 and HERVs plays a role in shaping, and perhaps enhancing, the T cell response to HIV-1. This justifies the consideration of HERV-K(HML-2)-specific and cross-reactive T cell responses in the natural control of HIV-1 infection and for exploring HERV-K(HML-2)-targeted HIV-1 vaccines and immunotherapeutics.