Conceived and designed the experiments: MA INC. Performed the experiments: MA. Analyzed the data: MA DJT INC. Contributed reagents/materials/analysis tools: WB. Wrote the paper: MA WJB DJT INC.
The authors have declared that no competing interests exist.
T lymphocytes of the CD8+ class are critical in delivering cytotoxic function and in controlling viral and intracellular infections. These cells are “helped” by T lymphocytes of the CD4+ class, which facilitate their activation, clonal expansion, full differentiation and the persistence of memory. In this study we investigated the impact of CD4+ T cells on the location of CD8+ T cells, using antibody-mediated CD4+ T cell depletion and imaging the antigen-driven redistribution of bioluminescent CD8+ T cells in living mice. We documented that CD4+ T cells influence the biodistribution of CD8+ T cells, favoring their localization to abdominal lymph nodes. Flow cytometric analysis revealed that this was associated with an increase in the expression of specific integrins. The presence of CD4+ T cells at the time of initial CD8+ T cell activation also influences their biodistribution in the memory phase. Based on these results, we propose the model that one of the functions of CD4+ T cell “help” is to program the homing potential of CD8+ T cells.
Immune responses against viruses and intracellular pathogens are often delivered by CD8+ cytotoxic effector cells, which kill the pathogen-infected cell and generally the pathogen too. However CD4+ “helper” T cells contribute to the function of CD8+ T cells in such infections. A striking example is found in Hepatitis C Virus infection, where the capacity of both humans and chimpanzees infected with the virus to achieve suppression of viremia is strongly correlated with the strength and diversity of the CD4+ T cell response
The mechanism of CD4+ T cell help in CD8+ T cell responses is complex, and incompletely understood. One important mechanism of action may be
After full activation, either through a licensed antigen-presenting cell or in the direct presence of CD4+ T cells, CD8+ T cells must make local contact with pathogen-infected host cells. This is critical since their cytotoxic effector function must be focused on the pathogen-infected target cell, with minimal engagement of other tissue cells. Thus, CD8+ T cells must recirculate and localize to sites of infection. The
In the present study, we addressed the significance of CD4+ T cell help in CD8+ T cell localization, using bioluminescent imaging of Luciferase-transgenic CD8+ T cells. The expression of a transgene encoding firefly Luciferase, and controlled by a strong T cell-specific promoter, allowed us to determine the biodistribution of CD8+ T cells in albino mice. In parallel, we labeled CD8+ T cells with the dye CFSE, and used it to reveal cells that had undergone multiple replication cycles, while co-staining for such cells for integrins and chemokine receptors allowed us to visualize the changes in the expression of such homing molecules that occur in dividing T cells. The use of a replication-defective virus-based vector allowed us to deposit the test antigen, ovalbumin, in skeletal muscle, while transgenic CD8+ T cells expressing an ovalbumin-specific antigen receptor made a local and systemic immune response. Antibody-mediated CD4+ T cell depletion allowed us to test the significance of “help”, delivered by such CD4+ T cells, in the CD8+ T cell response. We conclude that in addition to its other effects, CD4+ T cell help affects the biodistribution of CD8+ T cells responding to antigen.
To purpose of our study was to test the effect of CD4+ T cell help on the location of CD8+ T cells during an immune response to a replication-defective Herpes Simplex Virus (HSV) amplicon-based vector that encodes ovalbumin
Either anti-CD4-treated, or saline-treated control mice were given 1×106 units of HSV-based amplicon vector
In normal mice, bioluminescent signal was evident in various superficial lymph nodes, including the cervical nodes, upon which we focused since these nodes were readily identifiable, bilateral, and did not drain the vector injection site. In addition, there was strong bioluminescence that developed in the abdominal region (
(A) Mice were immunized with HSVova or HSVlac, and 24 hr later infused intravenously with 1×104 purified double transgenic naïve CD8+ T cells, and then imaged using an IVIS imager. The bioluminescent images represent the biodistribution of the CD8+ cells on day seven. The control group of mice had their endogenous CD4+ T cells present at the time of HSova immunization (left panel). The CD4 depleted group received three injection of anti-CD4 monoclonal antibody GK1.5 before they were immunized with HSVova (center panel). The MHC class II deficient mice lacking functional CD4+ T cells were similarly immunized (right panel). Living image software allows the superimposition of elliptical regions of interest, within which the photon flux was measured. (B) Time course of abdominal bioluminescence after administration of HSVova or HSVlac and 1×104 T-lux/OT-1 CD8+ cells. Data are representative of 6 to 12 individually analyzed mice, and are mean ± SEM.
Deficiency of CD4+ T cells affected not only the level of bioluminescence, but in particular its biodistribution, changing the signal to a lesser extent in the cervical lymph nodes, but more strikingly in the abdomen. Thus, anti-CD4+ antibody treatment attenuated the signal (
While the effect of CD4+ T cell deficiency on bioluminescent signal in the cervical lymph nodes was significant but partial (
An alternative explanation for the differential effects of CD4+ T cells in cervical
Activated OT-1 cells were obtained in all lymphoid tissue examined, and the pattern of proliferation of CD8+ antigen specific T cells from both positive control and CD4 depleted mice were similar (
(A) Mice that received CFSE-labeled naïve OT-1 CD8+ T cell were immunized i.m. with HSVova. Three days post adoptive transfer of ova specific CD8+ cells, the proliferation of CFSF-labeled cells in mesenteric (MLN), inguinal (ILN) and cervical lymph nodes (CLN) and from the spleen (SPL) was visualized using flow cytometry. All dot plots were gated on CD8+ T cells. (B) Cell numbers obtained from MLN, ILN, CLN and SPL at day three post adoptive transfer of ova specific OT-1 T cells. There were significantly fewer OT-1 cells in CD4-depleted mice in the MLN (** p<0.013) and the SPL (*p<0.005) but not the other lymph nodes. Data are representative of six independent experiments (n = 2 mice per group for each experiment).
Antigen engagement by naïve T cells results in changes in the expression of homing and adhesion molecules. In particular, activated T cells interacting with intestine-derived dendritic cells selectively increased their expression of alpha4-beta7 integrin
(A) Expression of alpha4-beta7 integrin (left) or the beta7 integrin chain (right) was assessed by flow cytometry on undivided (high CFSE) ova specific CD8+ T cells, and on cells that had undergone more than four divisions (low CFSE), taken from MLN and CLN from control mice. Integrin expression was revealed by staining with a PE-conjugated antibody on day 4-post immunization. (B) The expression of adhesion molecules was analyzed by flow cytometry on undivided and divided CD8+ OT-1 cells in CD4 depleted mice. Comparing (A) with (B), there was a clear difference in the modal expression of alpha4-beta7 integrin in the MLN.
Mice were immunized with HSVova i.m. and 24 hr later infused i.v. with CFSE labeled CD8+ T cells. Different lymphoid organs were taken out four days post HSVova. The level of beta7 -integrin expression was calculated as the geometrical mean fluorescence intensity (GMFI) and is presented as a function of undivided (0) and more than 4 divisions, based on CFSE dilution. Upregulation of beta7 integrin on CD8+ T cells from MLN was significant (p = 0.0022) in control mice but not in CD4+ depleted mice. Mice were from three experiments (n = 2 mice per group per experiment).
The level of expression of alpha4-beta7 integrin was calculated as the geometrical mean fluorescence intensity and is presented as a function of cell division for each individual mouse. In the MLN, there were increase in alpha4-beta7 integrin expression in both control and CD4+ treated mice, but the increase in GMFI was significantly greater in control mice (p = 0.0325). Data are from three experiments (n = 2 mice per group per experiment). A GMFI of >0 implies fluorescence intensity lower than the unstained control.
These data show that activated antigen specific CD8+ T cells acquired tissue specific homing molecules within the lymph nodes 4 days after ova inoculation. In control mice, the ability to re-localize into intra-abdominal lymphoid organs increased after several rounds of division, in parallel with the acquisition of tissue specific homing molecules such as the beta7 integrin chain, and the alpha4-beta7 integrin heterodimer. The ability of such CD8+ T cells to up-regulate these gut homing markers was impaired in CD4-deficient mice.
To test whether CD4+ T cells, present during priming, had effects on CD8+ T cell location that persisted in the memory phase, we re-challenged the experimental mice with SIINFEKL peptide 45 days after priming. Mice were imaged before, and then at time intervals after the secondary challenge with peptide, which was given IP on three constitutive days, each dose being 25 nmol of SIINFEKL diluted in 100 µl of saline. In the cervical lymph nodes, we did not observe clear effects of CD4+ T cell depletion on CD8+ T cell localization. Thus, the bioluminescent signal appeared at first to be attenuated (but this effect was not statistically significant, p = 0.248) in mice that received anti-CD4 antibody treatment on day 2 post SIINFEKL (day 49 post priming) but 2 days later, on day 52, it reached a level comparable to control mice given the HSVova vector, and was in excess of control mice that had received the HSVlac control vector (data not shown). Overall, the bioluminescent signal in the CLN was not significantly different between the anti-CD4 treated and the untreated control mice (p = 0.4).
In contrast to the lymph nodes, the effect of CD4+ T cell depletion during priming was striking during the recall responses in the abdominal lymphoid organs. In the HSVova positive control mice, the photon flux in the abdominal region peaked four day after challenge (day 51 post priming;
(A) Mice were re-challenged with 25 nM SIINFEKL peptide 45 days post priming and biodistribution of T-lux/OT.1 cells was determined using
The data presented here show that CD4+ T cells influence the biodistribution of CD8+ T cells. This was shown using HSV-based amplicon vectors, given IM into the quadriceps femoris, as the antigen delivery vehicle. The CD8+ T cells were double-transgenic, expressing both an antigen-specific T cell receptor that recognizes an ovalbumin-derived peptide, and firefly Luciferase
The localization of activated CD8+ T cells to the mesenteric nodes after several days in the immune response to a replication-defective vector challenge in the hind limb is unlikely to be due to migration of either antigen or antigen-presenting cells to these nodes. A recent study shows that a tracer dye given into the hind foot localizes first to the popliteal and the inguinal lymph nodes, and subsequently drains to the iliac nodes
This change in CD8+ T cell abundance in different tissue sites could have resulted from several mechanisms. We tested for, and excluded a potential mechanism based on differential T cell division in the cervical
The immune responses of CD8+ T cells are sometimes strictly CD4+ T cell help-dependent, and sometimes more-or-less CD4+ help-independent, based on the nature of the immune stimulus. Help-independent responses are typical of those to an infectious agent, such as the bacterium,
The interaction of CD4+ T cells with CD8+ T cells may be mediated via dendritic cells, which are central in naïve T cell activation under most circumstances. This model that explains this 3-cell interaction, termed “licensing”, was proposed more than 10 years ago
The role of CD4+ T cell help in generating memory CD8+ T cells also varies between different experimental models. Early reports suggested that CD4+ T help is essential for the generation of functional CD8+ T cell memory to most antigens
The concept that CD4+ T cell help for CD8+ T cells is mediated by licensing of dendritic cells implies the recognition of antigen that is presented to the CD4+ T cell by the dendritic cell. Thus, the antigen specificity of the CD4+ T cell would be crucial for this kind of help. Our CD4+ T cell depletion experiments did not distinguish between antigen-specific and non-specific help, and it may have been better not to exclude either. The role of antigen-specificity in CD4+ T cell help for CD8+ T cells is ambiguous; one study showed that the presence but not the specificity of CD4+ T cells was important in long-term CD8+ T cell memory
The observation that a brief antigen exposure could result in clonal expansion of naive CD8+ T cells, followed by effector functions and memory cells, gave rise to the concept of “programming”
After priming, activated T cells enter the circulation and migrate to many tissues, including secondary lymphoid organs and non-lymphoid tissue. Studies by Masopust and colleagues
On the basis of the data presented here, we speculate that CD4+ T cell help for CD8+ T cells entails an element that we propose to call “programming for location”. While we have observed this only in a vector-driven model, it may be a general feature of helper-dependent CD8+ T cell responses. If this model is correct, programming for location may contribute to why CD4+ T cell help is important in effective CD8+ T cell-mediated immunity, for example to pathogens such as Hepatitis C Virus, in which some individuals eradicate infection, others fail to do so, and the difference is closely associated with the presence or absence of a CD4+ T cell response.
All animal experiments were approved by the Institutional Animal Care and Use Committee.
C57BL/6J-Tyrc-2J/J albino mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and were kept in the University of Rochester SPF vivarium. MHC class II-deficient mice were obtained from Taconic Farms (Germantown, NY, USA) and were backcrossed three times to C57BL/6L-tyrc-2J/J in order to have MHC class II deficient mice with an albino coat. For
Mice were typed using the allotypic marker CD45.1 (clone A20) PE conjugated, TCR Valpha2 (clone B20.1) PE-conjugated, Vbeta5 (clone MR9-4) FITC conjugated and CD8α (clone 53-6.7) PerCP conjugated antibodies from BD PharMingen (San Jose, CA, USA). Data were acquired using a FACSCalibur® (Becton Dickinson Immunocytometry System, San Jose, CA, USA).
Antibodies to the murine alpha4-beta7 heterodimer (clone DATK32), and beta7 (clone M293) integrin chains were from BD Pharmingen (San Jose, CA, USA). Both integrin antibodies were PE-conjugated. Lymphoid organs were harvested on day three-post adoptive CD8+ T cell transfer; these were the inguinal lymph nodes (ILN), mesenteric plus lumbar nodes (MLN), cervical lymph nodes (CLN) and the spleen (SPL). These tissues were homogenized mechanically and cells re-suspended in Hanks Balanced Salt Solution (HBSS) containing 5% heat inactivated fetal bovine serum. Cell suspensions were counted, and incubated with Live/DEAD stain (Invitrogen, Eugene, OR, USA) before surface marker antibody staining. Subsequently, cells were washed with PBS and re-suspended in PBS for FACS analysis using a LSRII™(BD Biosciences, San Jose, CA, USA). Doublets were excluded based on a gate on forward height
Mice were anesthetized with intraperitoneally (i.p.) injection of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight) and then given an i.p. injection of D-luciferin (214 µg/g body weight; Xenogen-Caliper corp., Alameda, CA, USA). After 5 min, the mice were positioned in the imaging chamber (IVIS-100 series) for data collection. Briefly, the IVIS-100 series consists of a cooled CCD camera mounted on a light-tight chamber, a cryogenic unit for camera refrigeration and a computer for image collection and analysis. The acquisition time was 5 min both for the primary response and for the secondary challenge. Bioluminescence data were obtained using medium binning, for high resolution but at the cost of loss of some sensitivity. Relative intensities of emitted light were represented as pseudocolor images ranging from red (most intense) to blue (least intense). Gray scale photographs and the corresponding pseudocolor images were superimposed with Living Image (Xenogen-Caliper) and Igor (waveMetrics, Lake Oswego, OR, USA) image analysis software. Signal emitted by regions of interest (ROI) was measured and data were expressed as photo flux and quantified as photon sec−1 cm−2 sr−1. The steradian (sr) term refers to photons emitted from a unit solid angle of a sphere. Data were shown as mean ± SEM. The machine background was subtracted electronically both from the images and from the measurements of photon flux.
Helper-free herpes simplex virus type 1 (HSV-1) based amplicon vectors encoding the test antigen ovalbumin were assembled in the Center for Neural Development and Disease, Amplicon Vector Production Core at University of Rochester (Rochester, NY, USA). The HSV amplicon consists of the HSV-derived immediate-early 4/5 promoters, which drive the expression of the ova gene (termed HSVova) or control HSV amplicon expressing beta-galactosidase (termed HSVlac). The amplicon can transduce a variety of tissues including post-mitotic neurons
We previously established an in vivo imaging protocol in which the immune response of adoptively transferred, double transgenic CD8+ T cells (T-lux/OT-1) against ova was visualizes non-destructively after IM inoculation of HSV-based amplicons
To document the duration of antigen expression, purified transgenic CD8+ T cells were transferred IV either 1 or 13 days after HSVova injection. Measurement of bioluminescent signal from intra-abdominal structures at 1 day post immunization resulted in maximal photon flux in the abdominal region by day 5 post cell transfer. In contrast there was no signal above baseline in immunized mice that received T-lux/OT-1 CD8+ T cells 13 days after HSVova (data not shown). These data show that expression of the HSV-encoded Ag was transient, even without exogenous T cells. The disappearance of antigen within 13 days is consistent with a previous report using HSV amplicon expressing Luciferase, in which the maximum signal from IM inoculation of HSVlac was at 24 hr and it declined rapidly thereafter
Peripheral lymph node and spleen cells were harvested from either OT-1 or T-lux/OT-1 mice. A single cell suspension was prepared by mechanical homogenization of tissues, and red blood cells were removed using Lympholyte M (Cedarlane Laboratories, Burlington, NC, USA). Naive purified CD8+ T cells were obtained using negative selection
GK1.5 (anti-CD4) hybridoma was grown in BD serum-free medium, then transferred to BD CELLine culture system (BD biosciences, Sparks, MD). Culture supernatant was harvested and pooled for antibody production. Pooled supernatant was purified by 50% ammonium sulfate precipitation, and dialysis. Antibody titer was measured by ELISA, and the antibody was sterilized by filtration. CD4+ cell depletion was achieved by i.p. injection of 50 micrograms of purified GK1.5 in 0.1 ml of PBS, given on three constitutive days prior to immunization. The effectiveness of depletion was monitored by staining peripheral blood with a non-competing anti-CD4 mAb (clone RM4-4) and anti CD3 (Clone 145-2C11) using a FACSCalibur® flow cytometer and CellQuest software. Both antibodies were purchased from BD Pharmingen (San Jose, CA, USA).
The SIINFEKL peptide, corresponding to residues OVA 257–264, was purchased from New England Peptide (Fitchburg, MA, USA) and mice were re-challenged with daily i.p. injections of 25 nmol of peptide for 3 days.
Bioluminescence data were pooled from multiple experiments and expressed graphically as mean ± SEM. The Mann Whitney test was used to measure the significance of difference observed between the experimental groups. Statistical analysis was performed using Prism software (GraphPad Software, Inc., La Jolla, CA, USA).
The absence of CD4+ T cell help does not affect the up-regulation of the CCR9 chemokine receptor. CFSE versus CCR9 staining was used to identify OT-1 T cells that had divided 4 or more times. The geometric mean fluorescence index (GMFI) was increased in dividing T cells in all of the lymph nodes examined, but unlike the case for alpha4-beta7 integrin, there was no significance difference between intact positive control mice and mice that were CD4-depleted.
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We thank Dr Casey Weaver (University of Alabama at Birmingham) for access to the T-lux mice. Also Clark Burris and Louis Lotta (University of Rochester) for helper virus-free amplicon packaging, Dr. Jyh-Chiang Wang for useful information concerning GK1.5 purification, Dr. Joe Hollenbaugh for training in IV injection and Dr. Timothy Bushnell with LSRII ™ training.