TAG HIV Basic Science, Vaccines, and Cure Project Blog

CD4 T Cell Responses to Commensal Bacteria in the Gut

There has been a lot of attention given recently to the role of gut CD4 T cell depletion in HIV pathogenesis. Surprisingly, very little is known about which antigens are targeted by gut CD4 T cells; at least one study has reported evidence of memory CD4 T cell responses to candida albicans (the fungus that causes thrush) but an absence of the typical memory responses against opportunistic pathogens (also called “recall responses”) found in the blood. More recently, gut HIV-specific CD4 T cell responses have been detected in some elite controllers and in HIV-infected individuals showing robust CD4 T cell recovery in the gut on antiretroviral therapy (this latter data was presented at CROI by Satya Dandekar, see the second talk in this webcast). Studies in mice have suggested that there are likely be CD4 T cell responses to commensal bacteria in the gut, but there is little research addressing this question in humans. In a new paper in press at Clinical Immunology, Rawleigh Howe and colleagues from Cara Wilson’s group at the University of Colorado describe their initial efforts to fill this knowledge gap. 

Using flow cytometric techniques, the researchers were able to detect the presence of CD4 T cells making interferon gamma in response to stimulation with several gut commensal bacteria species (Enterobacter, E. coli, Enterococcus species) as well as to the pathogen, Salmonella typhimurium. CD4 T cells making IL-17 – Th17 cells – were also detected but at a much lower frequency. Bacteria-specific CD4 T cell responses could also be detected in the blood but at significantly lower levels; the difference in magnitude between gut and blood ranged from 8.5 to 19.5 fold. When responses to all four bacterial antigens were summed, the median frequency of interferon gamma-producing CD4 T cells was 0.24% in the gut compared to 0.02% in blood.

The researchers suggest that the CD4 T cell responses revealed in this study play a role in containing bacteria in the gut under normal conditions. Such a role would be consistent with recent studies indicating that T cell depletion can lead to systemic dissemination of gut bacteria (microbial translocation). Another implication of the data is that people with HIV infection may have altered CD4 T cell reactivity to commensal bacteria, and Cara Wilson’s group is addressing this possibility in ongoing studies.

Clinical Immunology

doi:10.1016/j.clim.2008.12.003   

Article in Press

Evidence for dendritic cell-dependent CD4+ T helper-1 type responses to commensal bacteria in normal human intestinal lamina propria

Rawleigh Howe a, Stephanie Dillon a, Lisa Rogers a, Martin McCarter b, Caleb Kelly a, Ricardo Gonzalez b, Nancy Madinger a and Cara C. Wilson a.

a Department of Medicine, University of Colorado Denver, Denver, Colorado 80045, USA

b Department of Surgery, University of Colorado Denver, Denver, Colorado 80045, USA

Received 23 September 2008; accepted 10 December 2008.  Available online 27 January 2009.

Abstract

Reactivity of lamina propria (LP) T cells to commensal bacteria has been demonstrated in animal models of inflammatory bowel disease (IBD) and in humans with IBD, but few studies have evaluated the function of such cells in normal individuals. LP mononuclear cells (LPMC) were disaggregated from healthy human intestinal tissue and cultured with heat-killed commensal and pathogenic bacteria. CD3+CD4+ IFN-γ-producing (Th1) cells reactive to commensal bacteria were demonstrated at frequencies ranging from 0.05 to 2.28% in LPMC. Bacteria-specific Th1 responses were inhibited by anti-HLA-DR antibodies and chloroquine exposure, were enriched in LP relative to peripheral blood, and expressed effector memory cell markers. Bacteria-specific CD4+ T cell proliferation in vitro was dependent on the presence of LP dendritic cells (DCs), which produced pro-inflammatory cytokines upon bacterial exposure. These results suggest that bacteria-reactive DCs and CD4+ T cells in normal LP have substantial pro-inflammatory potential that is revealed upon disaggregation in vitro.

CROI Webcasts, Abstracts & Posters Online

The annual Conference on Retroviruses & Opportunistic Infections (CROI) was held last week in Montreal. Although CROI has taken flak over the years for having strict registration policies, it also deserves credit for offering the most comprehensive web coverage of any conference. In addition to webcasts of every session except poster discussions, pdf files of poster abstracts are also rapidly placed online on the conference website.

Among the highlights this year were results from a phase 2B study of the microbicide PRO2000/5 gel which offered encouraging evidence that the product might prove efficacious in a large ongoing phase III efficacy trial. The phase 2B results showed around 30% efficacy and just failed to reach significance, but multiple analyses appeared consistent with the product having some protective effect. Results from the phase III trial are expected before the end of 2009. The presentation of the data was by Salim Karim and his talk is the fourth presentation in the webcast of the “HIV Transmission: Characteristics and Prevention” session.

In vaccines, Phil Johnson described experiments designed to establish whether AAV vectors could be used essentially as gene therapies. Johnson has produced AAV constructs that encode and persistently express monoclonal antibodies capable of neutralizing SIV, and he showed at CROI that this approach protected three animals from SIV infection in preliminary challenge experiments. David Watkins also presented results from a study in which macaques that received a multi-antigen SIV vaccine were challenged with a heterologous virus, SIVsmE660. The degree of control of both peak and set point viral loads was unusually robust. Both of these talks can be viewed in the webcast of the Wednesday vaccine symposium.

One area of concern highlighted by CROI is the accumulation of data showing that a significant subset of individuals receiving HIV treatment remain at risk for clinical illness due to suboptimal immune reconstitution (despite effective viral suppression). A low nadir CD4 count prior to starting treatment appears to be the single largest risk factor, but other variables such as age and thymic function also appear to be involved. Many studies have now reported that both the extent of immunodeficiency and the duration of time spent with low CD4 levels is linked to an increased risk of both AIDS and non-AIDS events compared individuals with good CD4 recovery on treatment. These findings should spur renewed efforts to find immune-based therapies that might be able to boost immune reconstitution and thereby reduce risk of illness in this population, but there was little sign of that at CROI. The only significant IBT results that were presented described the sad denouement to the protracted tale of interleukin-2, which failed to show any clinical efficacy in two very large randomized trials, ESPRIT and SILCAAT. Those presentations and the other limited oral abstract offerings on immune-based therapies can be viewed in the webcast of the Tuesday morning session.

Can T Cells Protect Against SIV Infection?

At the AIDS Vaccines Conference in Cape Town last fall, Louis Picker offered some solace to a chastened field when he presented encouraging results obtained with a novel vaccine strategy in the SIV/macaque model. The study has now been published online in Nature Medicine. Picker set out to design a vaccine strategy that would maintain a population of activated “effector memory” T cell responses against SIV, based on the observation that these cells traffic to mucosal tissues and may therefore offer a rapid, front-line defense against virus challenge. The rhesus cytomegalovirus (RhCMV) was selected as the vaccine vector due to its persistence  and maintenance of effector memory CMV-specific T cells. Picker and colleagues report in the paper that this approach induced robust SIV-specific CD4 and CD8 T cell responses of the desired effector memory phenotype. Over a year after receipt of the vaccine, animals showed approximately equivalent CD4 and CD8 T cell responses to SIV antigens, totaling, on average, 1.5% and 2.0% of blood memory T cells, respectively. Macaques that were already infected with RhCMV developed similar responses to those that were RhCMV-naïve, suggesting that vector-specific immunity did not impact the immunogenicity of the approach.

Having established that the RhCMV vector induced SIV-specific effector memory T cell responses, the researchers went on to subject 12 immunized animals and 16 controls to a series of repeated, low-dose challenges with SIVmac239. More than half the control animals developed progressive, systemic infection after the first or second challenge, with the final control acquiring infection after 12 exposures. Immunized macaques required a median of eight exposures and, most notably, four of these animals completely resisted systemic infection. Two of these apparently protected animals briefly displayed low-level viral loads of 60-80 copies after the first exposure while the other two never had detectable virus in plasma after 13 exposures. All four of these macaques showed evidence of at least transient infection because CD8 T cell responses developed to SIV antigens (Pol and Vif) that were not included in the vaccine. Several months after the first challenge, the four protected animals were depleted of CD8 T cells using a monoclonal antibody, but virus remained undetectable in plasma and cell-associated SIV DNA or RNA was not detected in peripheral blood and lymph node CD4 T cells. Picker and colleagues write that these findings suggest “infection was effectively contained locally, before dissemination and establishment of typical systemic infection.”

Among the eight vaccinated macaques that developed systemic infection, viral loads were not significantly different from controls. The researchers state that this is not surprising because of the low proliferative capacity of effector memory T cells (TEM) compared to central memory T cells (TCM). The implication is that T cell responses that might be protective against acquisition are not necessarily synonymous with T cell responses that contribute to post-infection control of viral replication. In the conclusion of the paper, Picker and colleagues offer the following thoughts regarding the import of their findings:

“It may also be possible to engineer HIV/AIDS vaccines that generate and maintain both TEM and TCM cell components, the latter serving as a second line of defense if the initial TEM cell barrier is breached. Such TEM cell or combination TEM-TCM cell vaccines will probably not provide absolute protection against all HIV exposure (against parenteral routes, for example). However, our data suggest that vaccine-generated T cell responses are able to do more than simply lower viral replication set points. Specifically, HIV/AIDS vaccines with a TEM component may have the ability to protect against the sexual transmission of HIV.”

Thus, while previous debates about the role of TEM vs. TCM in protection have generally been framed simply in terms one or the other being more important, this new data may offer a more nuanced perspective. However, it’s probably worth bearing in mind that this perspective is based on results in four macaques. Similar examples of apparent protection against SIV are rare; to my knowledge the best result previously reported in the literature involved “clearance” of a mucosal SIVmac251 challenge by five out of 11 macaques immunized with a NYVAC regimen.

Letter

Nature Medicine

Published online: 15 February 2009 | doi:10.1038/nm.1935

Effector memory T cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge

Scott G Hansen1, Cassandra Vieville1, Nathan Whizin1, Lia Coyne-Johnson1, Don C Siess1, Derek D Drummond1, Alfred W Legasse1, Michael K Axthelm1, Kelli Oswald2, Charles M Trubey2, Michael Piatak, Jr2, Jeffrey D Lifson2, Jay A Nelson1, Michael A Jarvis1 & Louis J Picker1

Abstract

The rapid onset of massive, systemic viral replication during primary HIV or simian immunodeficiency virus (SIV) infection and the immune evasion capabilities of these viruses pose fundamental problems for vaccines that depend upon initial viral replication to stimulate effector T cell expansion and differentiation1, 2, 3, 4, 5. We hypothesized that vaccines designed to maintain differentiated effector memory T cell (TEM cell) responses5, 6 at viral entry sites might improve efficacy by impairing viral replication at its earliest stage2, and we have therefore developed SIV protein-encoding vectors based on rhesus cytomegalovirus (RhCMV), the prototypical inducer of life-long TEM cell responses7, 8, 9. RhCMV vectors expressing SIV Gag, Rev-Tat-Nef and Env persistently infected rhesus macaques, regardless of preexisting RhCMV immunity, and primed and maintained robust, SIV-specific CD4+ and CD8+ TEM cell responses (characterized by coordinate tumor necrosis factor, interferon- and macrophage inflammatory protein-1expression, cytotoxic degranulation and accumulation at extralymphoid sites) in the absence of neutralizing antibodies. Compared to control rhesus macaques, these vaccinated rhesus macaques showed increased resistance to acquisition of progressive SIVmac239 infection upon repeated limiting-dose intrarectal challenge, including four macaques who controlled rectal mucosal infection without progressive systemic dissemination. These data suggest a new paradigm for AIDS vaccine development—vaccines capable of generating and maintaining HIV-specific TEM cells might decrease the incidence of HIV acquisition after sexual exposure.

A Role for B Cells in the Development of CD4 T Cell Memory

Two new papers converge in concluding that B cells can play an important role in sustaining CD4 T cell memory, via a mechanism that is independent of antibody production. One study involves genetically manipulated mice, the other looks at people with genetic disorders that impact B cell numbers and function. The data appear consistent with reports of an increased incidence of viral, bacterial, and fungal infections in individuals that have received B cell-depleting therapies. The human study also identifies possible differences between pathogens, as influenza-specific T cell memory did not appear to be impacted in the same way as responses to meningococcus. 

As Jason Whitmire and colleagues point out in their paper, these findings have implications for interpreting studies that use experimental B cell depletion as a means to evaluate the role of neutralizing antibodies in protecting against disease. Several such studies have been conducted in the SIV model, for example this report in J Virology from 2007. The authors of the paper write: “Our results suggest that antibodies are critical for protecting SIV-specific memory CD4 T helper cells from devastating destruction in the initial stages of infection. Thus, monkeys that produce anti-SIV antibodies by week 4 PI are able to live for extended periods because SIV-specific CD4 T helper cells can persist and maintain effective antiviral CD8 T-cell responses.” However, Whitmire et al found that the absence of B cells caused CD4 T cell responses to disappear with very similar kinetics (at the end of the primary response, when a memory CD4 T cell response would normally be expected to develop and persist). These new data may therefore offer an alternate explanation of the loss of CD4 T cell memory in the SIV study.  

Coincidentally, at the upcoming Conference on Retroviruses and Opportunistic Infections (CROI), which starts tomorrow in Montreal, there is a poster presentation regarding the impact of the B cell-depleting drug rituximab in people with HIV infection (poster #331, see below for the full reference and link to the CROI pocket program).

The Journal of Immunology, 2009, 182: 1868-1876.

doi:10.4049/jimmunol.0802501

Requirement of B Cells for Generating CD4+ T Cell Memory

Jason K. Whitmire2,*, Mary S. Asano, Susan M. Kaech, Surojit Sarkar, Lynn G. Hannum, Mark J. Shlomchik and Rafi Ahmed

* Department of Immunology & Microbial Science, The Scripps Research Institute, La Jolla, CA 92037;   Emory Vaccine Center and Department of Microbiology & Immunology, Emory University School of Medicine, Atlanta, GA 30322;   Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520; and   Department of Immunobiology and Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT 06510

B cells can influence T cell responses by directly presenting Ag or by secreting Ab that binds to Ag to form immunogenic complexes. Conflicting evidence suggests that persisting Ag-Ab complexes propagate long-term T cell memory; yet, other data indicate that memory cells can survive without specific Ag or MHC. In this study, the roles of B cells and Ag-Ab complexes in T cell responses to lymphocytic choriomeningitis virus (LCMV) infection were investigated using B cell-deficient or B cell-competent mice. Despite normal lymphocyte expansion after acute infection, B cell-deficient mice rapidly lost CD4+ T cell memory, but not CD8+ T cell memory, during the contraction phase. To determine whether Ag-Ab complexes sustain CD4+ T cell memory, T cell responses were followed in B cell-transgenic (mIg-Tg) mice that have B cells but neither LCMV-specific Ab nor LCMV-immune complex deposition. In contrast to B cell-deficient mice, mIg-Tg mice retained functional Th cell memory, indicating that B cells selectively preserve CD4+ T cell memory independently of immune complex formation. An in vivo consequence of losing CD4+ T cell memory was that B cell-deficient mice were unable to resolve chronic virus infection. These data implicate a B cell function other than Ab production that induces long-term protective immunity.

Blood First Edition Paper, prepublished online February 6, 2009; DOI 10.1182/blood-2008-08-171587.

Impaired maintenance of naturally acquired T cell memory to the meningococcus in individuals with B cell immunodeficiency

Begonia Morales-Aza, Sarah J Glennie*, Tomaz Pereira Garcez, Victoria Davenport, Sarah L Johnston, Neil A Williams, and Robert S Heyderman

Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, Bristol, United Kingdom

Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi

Department of Immunology, North Bristol NHS Trust, Bristol, United Kingdom

Faculty of Applied Sciences, University of West of England, Bristol, United Kingdom

The importance of T cells in the generation of antigen specific B cell immunity has been extensively described, but the role B cells play in shaping T cell memory is uncertain. In healthy individuals, exposure to Neisseria meningitidis in the upper respiratory tract is associated with the generation of memory T cells in the mucosal and systemic compartments. However, we demonstrate that in B cell deficient subjects with X-linked agammaglobulinemia (XLA), naturally-acquired T cell memory responses to meningococcal antigens are reduced compared with healthy controls. This difference is not seen in T cell memory to an obligate respiratory pathogen, influenza virus. Accordingly, we show that meningococcal antigens upregulate MHC class II, CD40, CD86/80 expression on mucosal and systemic associated B cells and that antigen presentation stimulates T cell proliferation. A similar reduction in N. meningitidis but not influenza antigen-specific T cell memory was seen in subjects with X-linked hyper IgM syndrome (X-HIM), implicating the interaction of CD40-CD40L in this process. Together, these data implicate B cells in the induction and maintenance of T cell memory to mucosal colonizing bacteria such as N. meningitidis, and highlight the importance of B cells beyond antibody production but as a target for immune reconstitution.

16^th Conference on Retrovirses & Opportunistic Infections, Montreal, Canada, February 8-11, Abstract #331

A Critical Role for B Cells in Control of HIV Revealed by Rituximab Therapy

Kuan-Hsiang Huang1, G Cooke2, E Thomson2, S Fidler2, J Main2, D Muir2, J Weber2, A Frater1, M McClure2, and P Klenerman1

1 Univ of Oxford, UK and 2Imperial Coll London, UK

CROI Pocket Program Online

Immune Recovery on Antiretroviral Therapy

A free access paper and accompanying commentary in Clinical Infectious Diseases address the issue of long-term immune recovery on antiretroviral therapy (ART). The paper describes results from AIDS Clinical Trials Group (ACTG) study 384, the largest and most detailed evaluation of the effects of ART on CD4 T cell counts and other immune parameters. The commentary by Elvin Geng and Steve Deeks provides an excellent overview of the findings.

ACTG 384 stratified participants using baseline CD4 count, and the most striking consequence of being below 50 cells/mm3 was the depletion of naïve CD4 T cells and the consequent skewing of the naïve-memory ratio. The strata used in the study were  <50, 51–200, 201–350, 351–500, and >500 cells/mm3, and the median baseline CD4+ naive‐memory cell ratio for individuals starting with a CD4 cell count <50 cells was 0.21 at baseline and increased to only 0.43 at week 144 – this was still lower than the median pre-treatment ratio for the higher CD4 strata. The lower CD4 strata also had the poorest improvement in CD4/CD8 ratio, and as Geng & Deeks note in their commentary “These two trends—a low naive‐memory cell ratio and a low CD4:CD8 ratio—have been seen both in those with untreated HIV infection and in the elderly (>75 years of age).” Conversely, individuals who started ART with CD4 counts >350 attained immune profiles that overlapped with HIV-negative individuals. The ACTG 384 data complement the findings recently reported by Rita Effros and colleagues regarding the premature aging of the immune system in HIV infection.

Geng & Deeks also offer some intriguing thoughts as to how the results may relate to the elevated risk of clinical disease that has been reported in individuals with a poor CD4 T cell recovery on ART:

“We believe that the conclusions reached by Robbins et al. might provide a mechanistic explanation for why some patients who receive HAART remain at risk of disease. Naive T cells are critical in both mounting an effective adaptive immune response against novel antigens and maintaining normal T cell homeostasis. Those naive T cells that survive untreated HIV infection are at least partially dysfunctional and have impaired proliferative capacity. It has recently been shown that chronic inflammation contributes to failure of naive T cell homeostasis, either by causing too much proliferation or by causing failure of naive T cells to proliferate in response to either homeostatic signals (which leads to lack of robust peripheral CD4 gains) or to novel antigens (which leads to disease). Tying this together, it is possible to construct a testable model in which those patients who initiate HAART late in their disease course have residual inflammation, suboptimal CD4+ T cell gains, skewed immunophenotypic profiles, persistent T cell dysfunction, and increased risk of all‐cause morbidity and mortality.”

Among the studies cited in support of this model is the recent Christine Bourgeois paper suggesting that naïve T cell depletion can lead to microbial translocation, which in turn contributes to ongoing immune activation and naïve T cell depletion. However, the commentary also cautions that: “determining the causal association among these factors will be a challenge, given the complexity of the human immune system and the lack of therapeutic interventions.” One important implication of the data is that the few therapies that might have the potential to increase naïve T cell levels (currently, IL-7 and human growth hormone derivatives) deserve urgent and careful evaluation in people with suboptimal immune recovery on ART, as there may be the potential for these approaches to offer significant clinical benefits.

Clinical Infectious Diseases 2009;48:362–364

DOI: 10.1086/595889

EDITORIAL COMMENTARY

CD4+ T Cell Recovery with Antiretroviral Therapy: More Than the Sum of the Parts

Elvin H. Geng and Steven G. Deeks

University of California, San Francisco

Clinical Infectious Diseases 2009;48:350–361

DOI: 10.1086/595888

HIV/AIDS

MAJOR ARTICLE

Incomplete Reconstitution of T Cell Subsets on Combination Antiretroviral Therapy in the AIDS Clinical Trials Group Protocol 384

Gregory K. Robbins,1 John G. Spritzler,2 Ellen S. Chan,2 David M. Asmuth,4 Rajesh T. Gandhi,1 Benigno A. Rodriguez,6 Gail Skowron,7 Paul R. Skolnik,3 Robert W. Shafer,5 Richard B. Pollard,4 and the AIDS Clinical Trials Group 384 Team

1Massachusetts General Hospital, Harvard Medical School, 2Harvard School of Public Health, and 3Boston University School of Medicine, Boston, Massachusetts; 4University of California–Davis Medical Center, Sacramento, and 5Stanford University Medical Center, Stanford, California; 6Case Western Reserve University School of Medicine, Cleveland, Ohio; and 7Roger Williams Medical Center, Providence, Rhode Island

Background. Initiation of combination antiretroviral therapy (ART) results in higher total CD4 cell counts, a surrogate for immune reconstitution. Whether the baseline CD4 cell count affects reconstitution of immune cell subsets has not been well characterized.

Methods. Using data from 978 patients (621 with comprehensive immunological assessments) from the AIDS [Acquired Immunodeficiency Syndrome] Clinical Trials Group protocol 384, a randomized trial of initial ART, we compared reconstitution of CD4+, CD4+ naive and memory, CD4+ activation, CD8+, CD8+ activation, B, and natural killer cells among patients in different baseline CD4+ strata. Reference ranges for T cell populations in control patients negative for human immunodeficiency virus (HIV) infection were calculated using data from AIDS Clinical Trials Group protocol A5113.

Results. Patients in the lower baseline CD4+ strata did not achieve total CD4+ cell counts similar to those of patients in the higher strata during 144 weeks of ART, although CD4+ cell count increases were similar. Ratios of CD4+ naive‐memory cell counts and CD4+:CD8+ cell counts remained significantly reduced in patients with lower baseline CD4+ cell counts (350 cells/mm3). These immune imbalances were most notable for those initiating ART with a baseline CD4+ cell count 200 cells/mm3, even after adjustment for baseline plasma HIV RNA levels.

Conclusions. After nearly 3 years of ART, T cell subsets in patients with baseline CD4+ cell counts >350 cells/mm3 achieved or approached the reference range those of control individuals without HIV infection. In contrast, patients who began ART with 350 CD4+ cells/mm3 generally did not regain normal CD4+ naive‐memory cell ratios. These results support current guidelines to start ART at a threshold of 350 cells/mm3 and suggest that there may be immunological benefits associated with initiating therapy at even higher CD4+ cell counts.