Tc1 effector diversity shows dissociated expression of granzyme B and interferon-γ in HIV infection
Kleen, Thomas O; Asaad, Roberta; Landry, Samuel Jb; Boehm, Bernhard Oc; Tary-Lehmann, Magdalena
From the Department of Pathology and the aCenter for AIDS research, Case Western Reserve University, Cleveland, Ohio, bDepartment of Biochemistry, Tulane University Health Science Center, New Orleans Louisiana, USA, and cUniversity Hospital of Ulm, Section of Endocrinology, Ulm, Germany.
Correspondence to M. Tary-Lehmann, BRB 928, Case Western Reserve University, 10900 Euclid Avenue, Cleveland OH 44106, USA.
Received: 12 February 2003; revised: 30 May 2003; accepted: 30 June 2003.
Objective: To examine antigen specific cytotoxic effector T cell diversity in HIV infected individuals.
Design: We used a panel of previously defined HLA class I-restricted HIV peptides to stimulate CD8 cells in freshly isolated peripheral blood mononuclear cells of HIV infected patients, to determine cognate killing via the perforin–granzyme pathway and inflammation induced by secretion of interferon (IFN)-γ.
Methods: ELISPOT assays were used to measure the secretion of granzyme B (GzB) and IFN-γ at single cell resolution.
Results: In all nine patients only approximately 20% of the peptides triggered a canonical Tc1 response with simultaneous release of GzB and IFN-γ. The majority of these peptides (approximately 80%) that elicited recall responses fell into the ‘single positive’ category with induction of either GzB or IFN-γ alone. The GzB positive cells did not produce interleukin (IL)-4 or IL-5.
Conclusion: The GzB positive but IFN-γ negative CD8 cells are programmed to induce apoptosis mediated killing without inflammation while the GzB negative and IFN-γ positive CD8 cells could mediate inflammation without killing. This Tc1 CD8 effector cell diversity and the understanding of these differentiation mechanisms may enable the precise implementation and fine-tuning of fundamentally different defense strategies against HIV and other infections.
Infection with HIV triggers a vigorous CD8 cell response with several percent of the CD8 cells initially staining positive with a single tetramer specificity . The primary immune response and CD8 cells in particular seem to play a critical role in controlling the virus [2–7] with the magnitude of the functional CD8 T cell response towards HIV correlating inversely with viral load and disease progression [8,9]. Various mechanisms have been implicated in the failure of this vigorous response to control the infection over time, thus leading to progressive immunodeficiency [10,11].
In chronic HIV infection, only a fraction of the tetramer positive CD8 cells co-express IFN- γ . An intriguing additional explanation, other than the lack of CD8 cell activity due to senescence or anergy resulting from the lack of CD4 cell help , might be the development of effector cell diversity within the CD8 T cell pool itself [13,14].
Classically, effector CD8 cells have been defined by their capability to lyse virus-infected cells and to produce high amounts of IFN-γ . Like CD4 cells, CD8 cells were subsequently found in subpopulations that express type 1 and type 2 cytokine profiles [16–21]. More recently, additional diversity was found within type 1 CD8 cells in as much that central and effector memory CD8 cells differ in IL-2 versus IFN-γ expression [13,22]. Even within type 1 CD8 effector cells evidence is emerging that discrete subpopulations maybe present which have varied capability to secrete IFN-γ and kill. In fact studies of Mossman et al. showed that dependent on different cytokine environments, naive murine T cell receptor (TCR)-transgenic CD8 cells are able to differentiate in vitro into effector cells that either produce IFN-γ and do not kill, or into CD8 cells that kill but do not produce IFN- γ or IL-4 . In mice, these different type 1 CD8 effector classes can also be induced in vivo via adjuvant-guided differentiation. Injection of several MHC class I restricted peptides in incomplete Freund's adjuvant induces CD8 cells that kill but do not produce IFN-γ. In contrast, injection of the same peptides in complete Freund's adjuvant induces IFN-γ producing CD8 cells that mediate delayed-type hypersensitivity but do not kill (P.V. Lehmann, personal communication).
CD8 cells can kill target cells via Fas–FasL interactions, or by utilizing perforin and granzyme B (GzB). Perforin and GzB molecules are constitutively expressed and stored in cytolytic granules by CD8 memory/effector cells . During antigen recognition, the CD8 cell releases the content of these granules towards the target cell. Perforin inserts pores into the target cell membrane through which GzB can penetrate to activate the apoptotic pathways. Evidence has emerged that GzB can also exert cytolytic activity in the absence of the perforin channel . This novel pathway involves high affinity binding of GzB to the mannose-6-phosphate/insulin-like growth factor II receptor on the target cell surface . Utilizing this receptor, GzB can penetrate the cells to activate the apoptotic cascade . These classical and novel cytolytic pathways of the key cytotoxin GzB have drawn interest in its secretion by CD8 cells. GzB secretion by CD8 T cells cannot be studied appropriately by intracytoplasmic staining. Cytotoxic molecules such as GzB and perforin are not expressed in naive CD8 T cells, but are constitutively expressed and present within preformed granules of every effector and memory CD8 T cell, as has been described for the presence of IFN-γ mRNA [26–30]. This means that every memory cell, regardless of epitope specificity and pathogen specificity, would show up as positive in any experiment that uses intracellular staining alone. This has the very important implication that measurements of only intracellular effector molecules would not lead to information about the in vivo effect of these molecules, because depending on the disease setting it would remain unclear if the molecules would have been ever secreted in order to achieve their effector function in the body itself. In addition certain disease settings can include secretory defects that prevent lytic granule exocytosis [31–34].
For this reason we intended to measure secreted effector molecules from peptide specific effector/memory T cells with the recently developed GzB ELISPOT assay . The 400 times higher sensitivity of this assay in monitoring cytotoxicity compared to the classic chromium release assay  provides a useful tool for better understanding the CD8 cell mediated immunity. The peptide binding groove of MHC Class I molecules is closed at both ends and accommodates peptides less than 12 amino acid long. However, systematic mapping of determinants frequently necessitated the use of longer peptides, typically 20-mers. Testing ideal length (9-mers) and 20-mer peptides, we provide evidence that there is a prevalent population of GzB secreting CD8 cells that do not produce IFN-γ or IL-4/IL-5 in HIV infection and hence should be capable of killing via the granzyme pathway while neither qualifying as Tc1 nor as Tc2.
Human peripheral blood mononuclear cell donors
Blood samples were obtained from normal volunteers and from HIV patients from the Special Immunity Unit at the University Hospitals (Cleveland, Ohio, USA). To reproduce the data of the initial assay, seven subjects were retested 5 weeks later. All patients were on highly active antiretroviral therapy (HAART) at the time of testing. The CD4 counts ranged from 400 to 1070 × 106 cells/l. All studies were performed under the approval of the Institutional Review Board for Human Investigation at the University Hospitals of Cleveland. None of the individuals tested was HLA-typed. Peripheral blood mononuclear cells (PBMC) were separated from heparinized blood collected in green-top tubes by centrifugation on Isoprep density gradients (Robbins Scientific, Sunnyvale, California, USA).
ELISPOT assays were performed as described previously  testing PBMC within 24 h of isolation; freeze-thawed cells were not tested. Briefly, ImmunoSpot plates P50 (Cellular Technology Limited, Cleveland, Ohio, USA) were coated overnight at 4°C with 50 μl capture antibody granzyme B GB-11 (4 μg/ml; Hoelzel Diagnostika, Köln, Germany), IFN-γ M700A-E (2 μg/ml; Endogen, Woburn, Massachusetts, USA), IL-4 8D4-8 (5 μg/ml PharMingen, San Diego, California, USA), and IL-5 TRFK5 (5 μg/ml PharMingen) respectively. The plates were blocked with bovine serum albumin (10 g/l in PBS; PBS–BSA) for 1 h and washed three times with PBS. PBMC were plated in complete RPMI medium (94% RPMI + 5% AB serum + 1% l-glutamine) at 1 × 105 per well (unless specified differently in the Results or figure legend). RPMI was from BioWhittaker (Walkersville, Maryland, USA) AB serum from Gemini Bioproducts (Calabasas, California, USA) and was heat inactivated at 56°C for 30 min. HIV-specific peptides at 10 μM or 10 μg/ml phytohemagglutinin (PHA; Sigma, St. Louis, Missouri, USA) were added. The cells were cultured for 24 h at 37°C to allow optimal secretion of GzB and IFN-γ and 48 h for IL-4 and IL-5. The plates were washed three times with PBS, three times with PBS–Tween (0.5%), and the secondary detection antibodies biotinylated anti-granzyme B (GB10, 3 μg/ml; Hoelzel Diagnostika, Köln, Germany), IFN-γ M701 (0.5 μg/ml; Endogen), IL-4 MP4-25D2 (2 μg/ml; PharMingen), and IL-5 JES1-5A10 (2 μg/ml; PharMingen) diluted in PBS–BSA–Tween were added. After incubation overnight at 4°C and four washes with PBS–Tween, Streptavidin–horseradish peroxidase (HRP) conjugate (Dako Corporation, Carpenteria, California, USA) was added at a 1 : 2000 dilution in PBS–BSA, incubated for 2 h at room temperature, and removed by washing three times with PBS–Tween and three times with phosphate-buffered saline (PBS). Spots were visualized using the HRP-substrate AEC (Pierce Pharmaceuticals, Rockford, Illinois, USA). The AEC stock solution was prepared by dissolving 10 mg AEC in 1 ml N, N-dimethyl formamide (Fischer Scientific, Fair Lawn, New Jersey, USA). For the actual development, 1 ml of this AEC stock solution was freshly diluted into 30 ml of 0.1 M sodium acetate buffer (pH 5.0), filtered (0.45 μm), and mixed with 15 μl H2O2. One-hundred microliters of that final solution was plated per well. The reaction was stopped by rinsing with distilled water when clear spots became visible macroscopically (10–45 min). The plates were air-dried overnight before subjecting them to image analysis on a Series 1 ImmunoSpot Image Analyzer (Cellular Technology, Cleveland, Ohio, USA) specifically designed for automated evaluation of ELISPOT results.
The HIV 8–9-mer peptides used (Interactiva, Ulm, Germany) were previously defined, class I-restricted epitopes with different HLA binding properties as described in detail in the HIV molecular Immunology Database [37,38] (http://hiv-web.lanl.gov). The 20-mer peptides with 10-amino acid overlaps spanning the entire gp120 sequence from strain 89.6 were a generous gift of S. Landry. Control wells contained medium with PBMC or effector cells alone. The peptide concentration of 10 μM was defined as optimal after titration in previous ELISPOT assays.
Cell separations and flow cytometric analysis
CD4 and CD8 cells were obtained by negative selection, by passing PBMC through affinity columns Human CD4+, CD8+ Subset Column Kit (R&D Systems, Minneapolis, Minnesota, USA). The efficacy of enrichment was controlled by FACS analysis. Flow cytometry was performed as described previously  using a Becton Dickinson FACScan and staining with labeled anti-CD4, anti-CD8, and anti-CD3 antibodies (PharMingen). Between 5000 and 10 000 live cells were analyzed per sample. The enrichment for the desired phenotype was between 87 and 94%. Antigen presenting cells (APC) were isolated from PBMC by negative selection using the T Cell Depletion Cocktail (RosetteSep; Stemcell Technologies, Vancouver, British Columbia, Canada).
HLA-Class I restricted HIV-peptides induce GzB and IFN-γ producing cells
In the first set of experiments, we tested PBMC of four healthy HIV negative donors and of nine HIV infected individuals. The latter had been HIV antibody positive for more than 2 years, were clinically stable and had CD4 cell counts between 400 × 106 and 1070 × 106 cells/l. All HIV infected individuals were on HAART therapy at the time of testing. PBMC (100 000 per well) were tested directly ex vivo by challenging for 24 h with a library of HIV peptides that consisted of 21 previously defined CD8 determinants [37,38] of HIV proteins gp120, nef, p17, pol, gp41 and p24 (the peptides are specified in Tables 1 and 2). For each tested peptide and patient, the secretion of GzB and of IFN-γ were measured in parallel. As shown in Table 1, seven of these nine HIV patients responded to at least one of the HIV peptides with GzB producing cells at a frequency > 5 : 100 000. Three patients responded to 4, 8 and 10 peptides, respectively. By adding up the frequencies of cells that responded to individual peptides (each reflecting the clonal size of cells responding to the individual peptide), we calculated the cumulative clonal mass of HIV reactive GzB producing cells for each patient (Table 1). This cumulative clonal mass of GzB producing cells was 46, 6, 22, 146, 12, 14 and 224 for the seven of the nine HIV patients who tested positive for at least one peptide (using the > 5 : 100 000 cut off criterion). In contrast, none of the four healthy controls responded to any of the peptides with > 5 : 100 000 GzB producing cells (Table 1). Thus while the numbers of positive peptides permits us to distinguish between infected and non-infected individuals, an even better distinction could be made by calculating the total clonal mass of peptide reactive GzB and IFN-γ producing cells.
In a second set of experiments, we tested seven HIV infected patients and four controls for responses to an overlapping series of gp120 peptides (47 peptides of 20 amino acids, progressing through the sequence in increments of 10 amino acids ). The individual responses of three representative patients are shown in Fig. 1a–c. All seven of the HIV infected individuals responded with GzB producing cells > 5 : 100 000 (Fig. 1d). Patients had minimum responses to at least three different peptides with a maximum response of up to 16 different peptides (Fig. 1b). This cumulative clonal mass of GzB producing cells was 335, 1273, 1899, 337, 181, 113 and 66 (Fig. 1d). Of the four healthy individuals two did not display any reactivity to the peptides tested, and two displayed minor reactivity (Fig. 1d). These latter control subjects (K and M) were health care workers who might have been environmentally sensitized to HIV [39–41]. They responded to few peptides (four and two, respectively) with low frequency GzB producing cells (< 46 per 100 000). The HIV infected individuals’ cumulative response (with the exception of patient I) were 2.5 to 41 times the mass of the highest responding control (control K). Overall a significant GzB reactivity to HIV peptides was seen only in HIV infected individuals.
IFN-γ measurements performed in parallel gave similar results (Fig. 1a–d). The HIV peptides did not elicit IFN-γ responses in non-infected controls including the health care workers who responded weakly with GzB release (Fig. 1d). Eight of the nine HIV patients tested mounted IFN- γ producing cells when challenged with the previously defined class I restricted HIV peptide library (Table 2) and seven of the seven patients tested with the gp120 peptide series (Fig. 1d) displayed reactivity to at least two peptides and maximally to as many as 20 peptides (Fig. 1a). The frequencies of the IFN-γ producing cells elicited by individual peptides were in the < 62 per 100 000 range, comparable to the frequencies of peptide-induced GzB producing cells. These results obtained measuring IFN-γ by ELISPOT is in agreement with published data [9,42].
HIV peptide-induced GzB producing cells are CD8
The HIV 8–9-mer peptides previously defined as T cell epitopes were class I-restricted with different HLA binding properties (Tables 1 and 2); they are described in detail in the HIV molecular Immunology Database (http://hiv-web.lanl.gov) [37,38]. In contrast, the 20-mer peptides used could trigger both CD4 and CD8 cell activity. We tested these 20-mer peptides in parallel on unpurified PBMC and on purified CD4 and CD8 cell fractions using autologous APC (T cell depleted PBMC). Fig. 1e and f show a representative experiment. The donor displayed responses to peptides 8, 30, and 47, but did not respond to peptide 37. Peptide 8 induced reactivity in CD4 cells only, peptides 30 and 47 induced reactivity in both the CD4 and the CD8 compartment (Fig. 1e). In contrast, all peptide-induced GzB production originated from CD8 cells (Fig. 1f).
HIV specific CD8 cells occur in GzB+/IFN-γ+, GzB+/IFN-γ−, and GzB−/IFN-γ+ subpopulations
Canonical Tc1 cells capable of GzB/perforin killing should coexpress IFN- γ. Indeed, approximately 18% of the 8–9-mer peptides that induced GzB production also triggered IFN-γ (Table 3). Also among the 20-mer peptides that triggered CD8 cells to produce GzB, approximately 27% elicited IFN-γ as well (Table 3). A representative example is shown in Fig. 2c. Strikingly however, 40% and approximately 33% respectively of the peptide-induced GzB-producing CD8 cells did not secrete detectable IFN-γ (Table 3). A representative example is shown in Fig. 2b. Some of the peptides elicited GzB producing cells at a frequency higher then 100 per 100 000 while triggering fewer than 5 per 100 000 IFN-γ producing cells (e.g., Fig. 1a, peptide 32). Because these measurements were carried out at single cell resolution , the low frequency of IFN-γ positive cells suggests that within some of these peptide responses the former were outnumbered up to several ten-fold by GzB+/IFN-γ− CD8 cells.
Among the 8–9-mer peptide induced CD8 cells, approximately 42% induced IFN-γ in the absence of detectable GzB (Table 3). For the 20-mer peptides this number was approximately 40% (Table 3 and Fig. 2a). The different GzB/IFN-γ phenotypes of the peptide-reactive cells were found to be stable with various peptide concentrations and when retesting the patients 5 weeks later (data not shown).
In separate experiments we tested whether the GzB+/IFN-γ− CD8 cells produce IL-4 or IL-5. Single cell resolution ELISPOT assays were performed to measure production of the latter cytokines. No triggered IL-4 or IL-5 responses were observed in GzB producing cells (data not shown). The GzB+/IFN-γ− CD8 cells therefore are not of a Tc2 phenotype. Since GzB+/IFN-γ− CD8 do not secret classic type 1 or type 2 cytokines, these cells seem to represent a novel lineage of CD8 effector cells.
Evidence is emerging that a greater effector T cell diversity exists than predicted by type 1/type 2 dichotomy cytokine expression patterns [12,13,43]. Type 1 and type 2 cytokines are mostly expressed in a mutually exclusive manner: frequently only one cytokine per T cell is expressed, and this expression even underlies allelic exclusion [22,44,45]. The data presented here suggest that in HIV, IFN-γ production by CD8 cells is linked with the ability to produce GzB only for approximately 20% of the peptides recognized. Therefore, only around 20% of the CD8 cells in HIV correspond to the classic Tc1 phenotype. We have identified two additional subpopulations that each occur at a higher frequency, constituting 30–40% of the HIV specific CD8 cell pool. One of these subpopulations secretes IFN-γ only. We propose to term this subpopulation Tc1b, as opposed to the classic Tc1 cell (Tc1a) (Table 4). Based on their secretion profile, Tc1b cells are pro-inflammatory: like Th1 CD4 cells, they do not kill but exert indirect effector functions by activating macrophages to engage in local delayed-type hypersensitivity reactions. In contrast, the CD8 subpopulation that secretes GzB only (Tc1c) can kill without inducing inflammation. Evidence for Tc1c mediated effector functions may have been observed in melanoma trials. Autoimmune, CD8 cell mediated destruction of normal melanocytes is a frequent side effect of this therapy, resulting in vitiligo-like depigmentation [46,47]. However, no signs of inflammation are seen in these lesions  and, strikingly, GzB and perforin were detected in T cells in the vicinity of disappearing melanocytes .
The GzB+/IFN-γ− and the GzB−/IFN-γ+ CD8 effector cell populations might represent separate effector cell lineages arising from instructed differentiation. These two phenotypes can be selectively engaged in vitro when naive TCR-tg CD8 cells are cultured in different cytokine microenvironments , and also by immunizing mice with MHC class I restricted peptides in different adjuvants (P.V. Lehmann, personal communication). It is unclear why within the CD8 cell response of individual HIV patients the GzB+/IFN-γ− and the GzB−/IFN-γ+ phenotype are segregated to different peptides. One possibility is that the CD8 cells secreting only GzB+ or only IFN-γ+ are primed in different microenvironments, for example in different tissues, or at different time points in the course of the disease, at stages when the net cytokine environment shifts.
It needs to be elucidated whether Tc1b or Tc1c cells are better suited to control HIV infection. While IFN-γ mediated inflammation is critical for host defense against many intracellular pathogens, including Mycobacterium tuberculosis, it is unclear whether it is also beneficial for controlling HIV. Macrophages are a primary target of HIV infection  and it has been shown that IFN-γ reactivates the expression of HIV in persistently infected promonocytic cells . Furthermore, IFN-γ upregulates the expression of the HIV coreceptor CCR5 in macrophages [52,53] and increases the susceptibility of these cells to infection with HIV-1 . Therefore, IFN-γ secreting Tc1a and Tc1b cells may have an adverse effect on the control of HIV replication. In contrast, GzB+/IFN-γ− CD8 cells (Tc1c) could be more suitable for controlling HIV infection. Tc1c effector cells are likely to destroy infected target cells including macrophages without activating viral replication in macrophages and without attracting additional susceptible cells into the vicinity.
Unlike the traditional chromium release assays, ELISPOT assays have high sensitivity that permits testing of large peptide libraries while using limited amount of PBMC [9,35,55–57] even on freeze-thawed material [42,58]. For these reasons, the trend has developed in the field to substitute 51Cr with IFN-γ ELISPOT assays [12,37,38,42,59,60]. This approach assumes that killing and IFN-γ production are linked effector functions. Also, monitoring of cellular immunity to HIV frequently relies on longer peptides, such as 20-mers. As seen in Table 3, 9-mer peptides and 20-mer peptides induced the IFN-γ/GzB single positive CD8 cells in similar proportions, that is 30–40% in each category. Therefore, the 9-mer and 20-mer peptides seem to stimulate the same subsets of CD8 cells. However, while the 20-mer peptides triggered IFN-γ production in CD4 and CD8 cells, they induced GzB only in the CD8 cell fraction. Long peptides, such as 20-mers preferentially bind to MHC Class II molecules (explaining the activation of IFN-γ producing CD4 cells). However, shorter peptide synthesis byproducts, or processed fragments of these 20-mers could trigger MHC Class I restricted CD8 cells. While CD4 and CD8 cells are capable of producing IFN-γ (and were therefore detected in IFN-γ assays), only CD8 cells are capable of producing GzB. IFN-γ assays using long peptides and unseparated PBMC are frequently used for CD8 cell monitoring. Our data clearly show that the IFN-γ assays with long peptides detect CD4 and CD8 cell immunity while GzB assays measure CD8 cell activity only.
The notion that we put forth here, that GzB positive but IFN-γ negative CD8 effector cells may constitute 35–40% of CD8 effector cells in response to HIV infection could be of utmost importance for CD8 cell monitoring in disease settings and vaccine trials. Our data suggest that measuring IFN-γ only will leave a substantial effector cell mass undetected, and perhaps even more importantly, ignore a CD8 cell type that is central to exerting protective immunity.
In summary, we show here that GzB+/IFN-γ− CD8 cells constitute a major effector cell class in HIV infection and that this cell type can be readily monitored ex vivo using GzB ELISPOT assays . Further studies of GzB+/IFN-γ− versus GzB−/IFN-γ+ CD8 cells in HIV and other infections should yield insights as to which of these is the class of response that affords higher protective value and thus should aid management of the infections and vaccine design.
We thank P.V. Lehmann, M. Lederman and D.D. Anthony for their valuable discussions.
Sponsorship: Supported by the NIH grant AI47756 and in part by the Center for AIDS Research at Case Western Reserve/University Hospitals of Cleveland (AI-36219) to M.T.L. and by NIH grant AI427202 to S.J.L. B.O.B. was supported by DFG SEB 518 and BMBF (IZKF-project A1).
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