Original Article

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Association between the HLA haplotype and the TCR-Vβ repertoire of anti-hCMV specific memory T-cells in immunocompetent healthy adults

Arancha Rodriguez-Caballero

Centro de Investigacion del Cancer/IBMCC (CSIC/USAL) and Servicio General de Citometria, Universidad de Salamanca, 37007 Salamanca, Spain

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Andres C. Garcia-Montero,

Andres C. Garcia-Montero

Centro de Investigacion del Cancer/IBMCC (CSIC/USAL) and Servicio General de Citometria, Universidad de Salamanca, 37007 Salamanca, Spain

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Julia Almeida,

Julia Almeida

Centro de Investigacion del Cancer/IBMCC (CSIC/USAL) and Servicio General de Citometria, Universidad de Salamanca, 37007 Salamanca, Spain

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Ana Balanzategui,

Ana Balanzategui

Servicio de Hematología, Hospital Universitario de Salamanca, 37007 Salamanca, Spain

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Santiago Munoz-Criado,

Santiago Munoz-Criado

Servicio de Microbiologia, Hospital Universitario de Salamanca, 37007 Salamanca, Spain

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Alberto Orfao,

Corresponding Author

Alberto Orfao

[email protected]

Centro de Investigacion del Cancer/IBMCC (CSIC/USAL) and Servicio General de Citometria, Universidad de Salamanca, 37007 Salamanca, Spain

Centro de Investigación del Cáncer and Servicio General de Citometria, Universidad de Salamanca. Campus Miguel de Unamuno. 37007 Salamanca, SpainSearch for more papers by this author

Arancha Rodriguez-Caballero,

Arancha Rodriguez-Caballero

Centro de Investigacion del Cancer/IBMCC (CSIC/USAL) and Servicio General de Citometria, Universidad de Salamanca, 37007 Salamanca, Spain

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Andres C. Garcia-Montero,

Andres C. Garcia-Montero

Centro de Investigacion del Cancer/IBMCC (CSIC/USAL) and Servicio General de Citometria, Universidad de Salamanca, 37007 Salamanca, Spain

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Julia Almeida,

Julia Almeida

Centro de Investigacion del Cancer/IBMCC (CSIC/USAL) and Servicio General de Citometria, Universidad de Salamanca, 37007 Salamanca, Spain

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Ana Balanzategui,

Ana Balanzategui

Servicio de Hematología, Hospital Universitario de Salamanca, 37007 Salamanca, Spain

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Santiago Munoz-Criado,

Santiago Munoz-Criado

Servicio de Microbiologia, Hospital Universitario de Salamanca, 37007 Salamanca, Spain

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Alberto Orfao,

Corresponding Author

Alberto Orfao

[email protected]

Centro de Investigacion del Cancer/IBMCC (CSIC/USAL) and Servicio General de Citometria, Universidad de Salamanca, 37007 Salamanca, Spain

Centro de Investigación del Cáncer and Servicio General de Citometria, Universidad de Salamanca. Campus Miguel de Unamuno. 37007 Salamanca, SpainSearch for more papers by this author

First published: 14 August 2007

https://doi.org/10.1002/cyto.b.20187

Citations: 7

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Abstract

Background:

Despite the key role of memory T-cells specific for human cytomegalovirus (hCMV) in protecting against hCMV-reinfection early after immunodeficiency episodes, the precise characterization and definition of the essential components of a protective CD4 T-cell response still remain to be established.

Methods:

We analyzed by flow cytometry hCMV-specific immune responses driven by peripheral blood antigen-presenting cells (APC) and CD4 memory T-cells at both the cellular and soluble levels, and their cooperation in priming and sustaining the effector function of specific CD8 T cells in adult healthy individuals using a hCMV whole viral lysate stimulatory model.

Results:

Overall, activated T-cells showed a heterogeneous phenotype, with a marked predominance of CD45RA⁻/CCR7^+/− memory CD4⁺ T-cells. Despite this, cytoplasmic expression of granzyme B was found in both the CD45RA⁺/effector and CD45RA⁻/memory T-cell compartments of the two major CD4⁺ and CD8⁺ activated T-cell subpopulations, further confirming the presence of circulating antigen experienced cytotoxic CD4⁺ T cells in hCMV-seropositive individuals. Moreover, we observed that both CD4⁺ and CD8⁺ hCMV-specific T-cells included relatively restricted numbers of TCR-Vβ family members. Interestingly, we found a significant association between some HLA Class II and Class I haplotypes and the presence of specifically expanded TCR-Vβ clones of anti-hCMV T cells.

Conclusions:

These results indicate that hCMV-specific memory T-cells are phenotypically heterogeneous, their TCR-Vβ repertoire shaped through the interaction between hCMV epitopes and the HLA haplotype. © 2007 Clinical Cytometry Society.

Cytomegalovirus (CMV) infection is a paradigm for the persistent viruses that actively manipulate and exploit the immune response (1). After primary infection, CMV remains in a latent state and episodes of viral reactivation are usually detected in blood only in immunocompromised patients (2, 3). In fact, the nonproductive state of CMV latency is associated with active cellular immune-surveillance, which rapidly leads to productive viral replication after withdrawal of T-and NK-cells (4). Accordingly, it has been shown that T-cell mediated immunity plays a key role in controlling CMV infection after immunodeficiency episodes (5).

For several years, attempts have been made to characterize CMV-specific T-cells. Recently, identification of CMV-specific CD8⁺ cytotoxic T-lymphocytes (CTL) as the main effector cells in CMV infection has greatly advanced through the use of peptide-loaded HLA-I tetramers (6-9). Although the presence of functional CD8⁺ T cells is essential to keep the virus in check during chronic infection, CD4⁺ T-cells are required to maintain an effective CTL response (10-12). Furthermore, the development of disease symptoms in CMV-infected patients has been associated with a delayed effector/memory CD4⁺ T-cell response (13). This suggests that functional effector CD8⁺ T-cells and antibody-mediated responses are not sufficient for controlling CMV infection and that formation of effector CD4⁺ T cells is essential. However, CD4⁺ T-cells are composed of heterogeneous subsets with different functional behavior, including CD4⁺ T cells with lytic capacity, which are detectable in the peripheral blood (PB) of CMV-infected subjects (14, 15). Despite this, information about the precise phenotypic characteristics and the role of these and other CD4⁺ T-cells in protecting against CMV as well as the definition of the essential components of a protective CD4⁺ T-cell response still remain a challenge.

In the present study we identified and characterized subpopulations of CMV-specific T-cells circulating in the PB of seropositive adult healthy individuals, particularly those T-cell subsets involved in the recognition of the virus in antigen presenting-cells (APC). Our results show the usage of an HLA-II haplotype-dependent restricted TCR-Vβ repertoire by CMV-specific CD4⁺ T cells displaying a heterogeneous Th1 memory/effector phenotype.

METHODS

Subjects and Samples

Heparin-anticoagulated PB samples from 28 adult volunteers (mean age of 30 ± 5 years; 11 males and 17 females) were studied. According to the hCMV serological status, as measured by the presence of anti-CMV specific IgG and IgM antibodies (LIAISON®CMV; DiaSorin, Saluggia, Italy), individuals were classified as being either CMV-seropositive (n = 18) or CMV-seronegative (n = 10). All individuals gave their informed consent prior to entering the study according to the recommendations of the local Ethics Committee.

Stimulation of PB Samples with hCMV

PB samples were diluted (1:1; vol:vol) in RPMI 1640 and cultured for 6h at 37°C in a 5% CO₂ sterile environment in the presence of 5 μg/ml of hCMV whole lysate (Advanced Biotechnologies, Columbia, MD), 30 μM of TAPI-2 (a tumor necrosis factor-alpha protease inhibitor; Cytognos, Salamanca, Spain) and 1 μg/ml of both the anti-CD28 and anti-CD49d costimulatory monoclonal antibodies (mAb) (Becton/Dickinson Biosciences -BDB-, San José, CA). An aliquot of each PB sample was processed in parallel in the absence of hCMV whole lysate, as a negative control-unstimulated sample.

Simultaneous Identification of hCMV-specific T-cells and Evaluation of Cytokine Secretion

The identification of hCMV-specific activated T-cells and the quantitative evaluation of the cytokines secreted in response to hCMV was performed as previously described in detail (16). Activated T-cells were defined as being CD3⁺, SSC^low, and TNF-α⁺. Specific quantification of IFN-γ, TNF-α, IL10, IL6, IL4, and IL2 was performed using the Human Th1/Th2 kit II CBA™ (BDB). Information about between 0.5 and 1.5 × 10⁵ events/sample aliquot was collected using a FACSCalibur flow cytometer and the CellQUEST software program (BDB). For data analysis, the PAINT-A-GATE PRO and CBA software programs (BDB) were used, as previously described (16).

Immunophenotypic Characterization of hCMV-specific TNF-α⁺ T-cells

In 17 CMV-seropositive samples, further immunophenotypic analysis of TNF-α⁺ T-cells was performed after stimulation with hCMV lysate for 6 h under the conditions described above, except for the fact that 10 μg/ml brefeldin A was added to one aliquot of the hCMV-stimulated and unstimulated samples, 2 h after the incubation had started. Once this incubation period was finished, samples cultured in the absence of brefeldin A were stained according to well described methods (17), with the following combination of mAb: CD45RA-FITC (clone RP1/11.1, ImmunoSTEP), TNF-α-PE (clone mAb11, BDB), CCR7-APCy (clone 150503, R&D Systems), CD3-PECy7 (clone SK7, BDB) and CD8-PerCP (clone SK1, BDB). Samples containing brefeldin A were sequentially incubated with CD45RA-FITC, TNF-α-APCy (clone 6401.1111, BDB), CD8-PerCP and CD3-PECy7 for the detection of cell surface antigens and anti-granzyme B-PE (clone GB-11; PeliCluster, Amsterdam; The Netherlands) for the detection of intracellular antigens. For this purpose the Fix & Perm reagent kit (Caltag Laboratories, San Francisco, CA) was used, as previously described (17). Information about between 5 × 10⁵ and 2 × 10⁶ events/sample aliquot was collected using a FACSAria flow cytometer (BDB). The FACSDiva software (BDB) program was used for both data acquisition and analysis. The gating strategy used for the identification and phenotypic characterization of TNF-α⁺/CD8⁻ and TNF-α⁺/CD8⁺ PB T-cells is illustrated in Figure 1.

Details are in the caption following the image — **Figure 1**
Open in figure viewer PowerPoint

Representative bivariate dot plots showing the immunophenotypic characteristics of hCMV-specific TNFα⁺ T-cells in a CMV-seropositive donor. Panels A to C illustrate how CD3⁺/TNFα⁺ T cells in response to hCMV lysate were sequentially identified and selected within the CD8⁺ and CD8⁻ T cell compartments. In panels D and E the pattern of expression of surface CCR7 and CD45RA as well as that of cytoplasmic _[c]granzyme B are shown for both TNFα⁺/CD8⁺ (blue dots) and TNFα⁺/CD8⁻ (red dots) T cell subsets. Of note, in the PB of this individual, part of the TNFα⁺/CD8⁻ T-cells showed a peripheral memory phenotype (CCR7⁻/CD45RA⁻/_cgranzyme B⁻) while activated TNFα⁺/CD8⁺ T-cells mainly showed phenotypic characteristics of effector/cytotoxic T-cells (CCR7⁻/CD45RA⁺/granzyme B⁺).

Analysis of the TCR-Vβ Repertoire of hCMV-specific Activated (TNF-α⁺) T-cells

Eight different aliquots of each PB sample corresponding to hCMV-seropositive subjects (300 μl/aliquot) were stimulated with hCMV-whole lysate as described above. Once stimulated, cells were stained for a total of 24 members of 21 different TCR-Vβ families using the TCR-Vβ repertoire Kit (Immunotech), following the recommendations of the manufacturer. For each staining, the following mAb were used in addition to the TCR-Vβ-FITC and TCR-Vβ-PE markers: CD3-PECy7, CD4-PerCPCy5.5 (clone SK3, BDB), and TNF-α-APCy. Information about between 1 and 2 × 10⁶ events/sample aliquot was collected using a FACSAria flow cytometer. The FACSDiva software was used for both data collection and analysis. A TCR-Vβ family member of CMV-specific T-cells was considered to be expanded in a PB sample once its percentage among the hCMV-reactive CD4⁺ or CD8⁺ T-cells was higher than the mean plus two standard deviations (SD) of the percentage of positive cells for the same TCR-Vβ mAb, within the total PB CD4⁺ or CD8⁺ T-cells present in the same sample, respectively.

DNA Preparation and HLA Typing

DNA was extracted from PB cells by the MagNAPure LC DNA Isolation kit (Roche Diagnostics GmbH, Mannheim, Germany). HLA-A and B alleles were identified by sequence based typing (SBT) methods (Applied Biosystems, Foster City, CA). Class II alleles (HLADRB₁ and HLADQB₁) and HLA-C were determined as follows: 1) for low resolution (antigen level) by sequence specific oligonucleotide probes-PCR (SSOP-PCR) using the INNO-LIPA HLA assay (INNOGENETICS NV, Ghent, Belgium) and 2) for high resolution (allele level) by sequence specific primer-PCR (SSP-PCR) using the Olerup SSP™ kit (Genovision, West Chester, PA) and the Dynal AllSet™ SSP kit (Dynal Biotech, Oslo, Norway).

Statistical Methods

Mean values and their SD, median and range were calculated for each variable under study, using the SPSS software (version 10.0; SPSS, Chicago, IL). In order to establish the statistical significance of the differences observed between groups, the nonparametric Friedman and Wilcoxon tests or the nonparametric Mann–Whitney U-test were used for paired and unpaired samples, respectively (SPSS 10.0 software). To analyze the degree of correlation between two variables, the lineal regression model was used (SPSS 10.0 software). Finally, statistically significant associations between TCR-Vβ expansions of hCMV-specific TNF-α⁺ T-cells and individual haplotypes were analyzed using the Pearson's χ² or the Fisher's exact tests, depending on the number of expected cases in each group. P-values <0.05 were considered to be associated with statistical significance.

RESULTS

Monocytic and T-cell Immune Responses to hCMV lysate

Immune responses to hCMV lysate were pronounced in CMV-seropositive individuals (n = 18), while almost undetectable in CMV-seronegative subjects (n = 10) (Fig. 2). Accordingly, after stimulation with hCMV whole lysate, the percentage of hCMV-activated (TNF-α⁺)/CD8⁻ (P = 0.001) and TNF-α⁺/CD8⁺ (P < 0.001) T-cells as well as that of TNF-α⁺ monocytes (P < 0.001) were significantly higher in CMV-seropositive than in CMV-seronegative donors (Fig. 2A). In addition, the soluble concentrations of secreted IFN-γ (P < 0.001), TNF-α (P < 0.001), IL-6 (P = 0.01) and IL-2 (P = 0.001) were also significantly increased in CMV-seropositive cases (Fig. 2B).

As expected, a significant correlation was found between the amount of TNF-α and IFN-γ secreted and the number of TNF-α⁺/CD8⁻ and TNF-α⁺/CD8⁺activated T-cells, respectively (Table 1). In turn, the overall amount of secreted IL2 showed a significant correlation with the number of TNF-α⁺/CD8⁺ activated T-cells but not with the number of TNF-α⁺/CD8⁻ T lymphocytes (Table 1).

Table 1. Correlation Between the Percentage of In Vitro Activated T-Cells and the Amount of Soluble Cytokines Produced in Response to hCMV Whole Lysate

Relationship Between the Anti-hCMV Inflammatory and T-cell Immune Responses

The number of TNF-α⁺ monocytes showed a correlation with the concentration of soluble IL-2 and TNF-α. In turn, a significant correlation was found between the amount of IL-6 secreted and both the number of TNF-α⁺/CD8⁻ T-cells and the soluble amounts of TNF-α secreted.

Immunophenotypic Characteristics of Anti-hCMV Specific TNF-α⁺ T-cells

Overall, most TNF-α⁺/CD8⁻ T-cells (81% ± 23%) showed a CD45RA⁻/CCR7^+/− memory phenotype, while a significantly (P = 0.001) smaller percentage (19% ± 23%) were CD45RA⁺/CCR7⁻ effector CD8⁻ T-cells (Fig. 3A). In contrast, within the TNF-α⁺/CD8⁺ T-cell population, similar percentages of CD45RA⁻/CCR7^+/− memory and CD45RA⁺/CCR7⁻ effector T-cells were observed (Fig. 3A). As expected, activated (TNF-α⁺) CD45RA⁺/CCR7⁻ effector T-cells showed a higher expression of cytoplasmic granzyme B in comparison to the TNF-α⁺/CD45RA⁻/CCR7^+/− memory T-cells both within the CD8⁻ (P = 0.01) and the CD8⁺ T-cell compartments (P = 0.02) (Fig. 3B).

The TCR-Vβ Repertoire of Anti-hCMV Specific TNF-α⁺ T-cells

A marked bias of the TCR Vβ repertoire was noted among hCMV-reactive TNF-α⁺/CD4⁺ and TNF-α⁺/CD8⁺ T-cells in all hCMV-seropositive donors analyzed (n = 16). Interestingly, 12 of the 24 TCR Vβ family members tested were significantly expanded in the activated TNFα⁺/CD4⁺ and/or TNFα⁺/CD8⁺ T-cell compartments, in response to hCMV (Table 2). Some TCR Vβ family members were expanded in both compartments of CD4⁺ and of CD8⁺ T-cells (Vβ2, Vβ4, Vβ5.1, Vβ13.6, Vβ14, and Vβ23) while a few were restricted to either TNF-α⁺/CD4⁺ (Vβ8, Vβ13.1, Vβ16, Vβ21.3, and Vβ22) or TNF-α⁺/CD8⁺ T-cells (Vβ17). The frequency at which each TCRβ family member was found to be expanded was variable within both the TNF-α⁺/CD4⁺ and the TNF-α⁺/CD8⁺ T-cells (Table 2).

Table 2. Expanded TCR-Vβ Families Within the TNF-α⁺/CD4⁺ and TNF-α⁺/CD8⁺ hCMV-Specific T-Cell Compartments and the Magnitude of Their Expansions

TCR-Vβ family		T-cell subset	Case number																% of cases with TCR-Vβ expansion
			1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16
% of TCR-Vβ⁺ CMV-specificTNFα⁺ T-cells	2	CD4⁺		24	21			42	32	27	33	22	49	46	22	18	30	30	81
	2	CD8⁺					5		17	7	22	37	20				33	20	50
	4	CD4⁺		4						9		5			13	3			31
		CD8⁺						3		19	14		48					4	31
	5.1	CD4⁺	14		15	14		10	9	19	11		17	20			12		63
	5.1	CD8⁺		51	41				28	20	13		33						38
	8	CD4⁺	8		8			11	8	7	14	11	7	14	11	7	13	6	81
	8	CD8⁺																	0
	13.1	CD4⁺		15	15	65	33												25
	13.1	CD8⁺																	0
	13.6	CD4⁺			5		29	9	9			4						5	38
	13.6	CD8⁺						5	4		1							7	25
	14	CD4⁺		8	8												6	8	25
	14	CD8⁺	27	18			58		9	15							33	9	44
	16	CD4⁺	7					2							2				19
	16	CD8⁺																	0
	17	CD4⁺																	0
	17	CD8⁺			37		37		24	11	13	11						22	44
	21.3	CD4⁺		3	4			5			7	3	6				7	5	50
	21.3	CD8⁺																	0
	22	CD4⁺	8	6	14		8	12	14	8	22	17	11	20		37	8	6	88
	22	CD8⁺																	0
	23	CD4⁺	11																6
	23	CD8⁺	28	12								52							19
	% expandedcells/totalTNFα⁺ cells	CD4⁺	48	60	90	79	70	91	72	70	87	62	90	100	48	65	76	60
	% expandedcells/totalTNFα⁺ cells	CD8⁺	55	81	78	0	100	8	82	72	63	100	100	0	0	0	66	62

Results are expressed as percentage of TCR-Vβ T-cell expansions within the hCMV activated (TNFα⁺) CD4⁺ and CD8⁺ T-cells. A specific TCR-Vβ T-cell subpopulation was considered to be expanded when its percentage among hCMV-activated (TNFα⁺) CD4⁺ and CD8⁺ T-cells was higher than the mean plus two standard deviations of the percentage of the same TCR-Vbeta family member within the total CD4⁺ and CD8⁺ T-cells. In the right column, the percentage of donors that had a specific TCR-Vβ T-cell expansion is shown for both the CD4⁺ and CD8⁺ T-cell compartments. In turn, the two lines in the bottom of the table show the sum of the percentages of the expanded TCR-Vβ T-cell families within the hCMV activated (TNFα⁺) CD4⁺ and CD8⁺ T-cells for each seropositive donor (n = 16).

In turn, the number of TCR-Vβ expansions observed among anti-hCMV specific T-cells from each CMV-seropositive subject analyzed varied notably. Accordingly, while in some cases (i.e., cases 3 and 6 from Table 2) numerous expansions of different TCR Vβ families were detected within hCMV-reactive CD4⁺ T-cells, (eight and seven different TCR Vβ family members, respectively), in others (i.e., cases 4 and 5) only a few (two and three TCR Vβ expansions within hCMV-specific TNF-α⁺/CD4⁺ T cells, respectively) were identified (mean number of 5 ± 2 expanded TCR Vβ family members/individual). Within the TNF-α⁺/CD8⁺ T-cell compartment a lower (P=0.002) number of TCR Vβ expansions per individual (mean 3 ± 2) was found in comparison to that of TNF-α⁺/CD4⁺ T-cells, ranging from zero (cases 4, 12, 13, and 14) to five expanded TCR Vβ family members (cases 7, 8, and 9).

Association Between the TCR-Vβ Expanded Family Members Within hCMV-specific TNF-α⁺ T-cells and the HLA Haplotype

A significant association between the TCR-Vβ repertoire of hCMV-specific CD4⁺ T-cells and the HLA Class II genotype was observed (Fig. 4). Accordingly, all four donors who showed expansion of TCR-Vβ13.1⁺/CD4⁺ T cells shared HLA-DRB₁*0701 (P = 0.001) and HLA-DQB₁*0202 (P = 0.001) haplotypes. In turn, an association between the expansion of TCR-Vβ16⁺/CD4⁺ T cells, observed in three individuals, and the HLA-DQB₁*0402 haplotype (P = 0.03), was also found. Likewise, a significant association between the TCR-Vβ repertoire of CMV-specific CD8⁺ T-cells and the HLA-I genotype was also observed for the HLA-C1203 allele and both TCR Vβ4 (P = 0.02) and TCR Vβ5.1 (P = 0.04), although a small proportion of cases showing expansion of these TCR Vβ family members within hCMV-reactive CD8⁺ PB T-cells (15% and 23%, respectively), did not have an HLA-C1203 haplotype.

DISCUSSION

The T-cell memory pool is finite in size (18), but the exact mechanisms by which memory cells are deleted still remain unclear. Previous studies indicate that antigens may play a critical role in this homeostatic selection protecting memory cells from terminal attrition (19). Thus, viruses such as hCMV, that persist in the host organism for long periods of time contribute to the establishment of clonal expansions of antigen-specific T-cells, commonly described within the CD8⁺ memory T-cell compartment (20). In contrast, CD4⁺ T-cell clones are less frequently detected (21) because they would require a sustained antigenic stimulation over years in order to expand (22). In the present study, we combined the use of a hCMV whole viral lysate stimulatory model in whole blood samples with that of a previously described (16) sensitive method for simultaneously evaluating specific immune responses by APC and T-cells at both the cellular and soluble levels. Since processing of viral lysate peptides is preferentially carried out via the exogenous pathway of antigen presentation on HLA-II molecules (23-25), such an approach would generally make it possible to identify and characterize CD4⁺ memory T cells.

Accordingly, as expected, we observed a predominant activation of hCMV-specific CD4⁺ T-cells. However, activation of hCMV-specific CD8⁺ T-cells was also found in most seropositive individuals. Such phenomena could be due to changes in APC induced by their interaction with CD4⁺ T-cells, allowing for cross-presentation of exogenous antigens and the induction of CD8⁺ T-cell responses [cross-priming] (26), which in turn, could be facilitated by secretion of soluble cytokines (27). Interestingly, both hCMV-specific TNF-α⁺/CD4 T-cells and TNF-α⁺ monocytes were significantly associated with the overall amount of TNF-α and IL-6 secreted; in contrast, although the amount of IL-2 secreted by T-cells in response to hCMV showed a significant correlation with the number of activated TNF-α⁺/CD8⁺ T-cells, it did not correlate with the number of CMV-specific activated TNF-α⁺/CD4 T-cells. Overall, this observation suggests the occurrence of varying patterns of T-cell responses in hCMV seropositive individuals; in addition, it supports the notion that IL-2 might play an important role in sustaining the in vitro effector function of hCMV-specific CD8⁺ T cells (28).

In order to gain further insight into the heterogeneity of hCMV-specific T cells, we characterized their phenotype in more detail. Overall, TNF-α⁺ T-cells showed a heterogeneous phenotype both within the CD4⁺ and CD8⁺ T-cell compartments. Accordingly, hCMV-specific TNF-α⁺/CD8⁺ T-cells showed a marked predominance of CD45RA⁺/CCR7⁻ effector cells as compared to TNFα⁺CD4⁺ T-lymphocytes, where a clear predominance of CD45RA⁻/memory T-cells was observed. Despite this, cytoplasmic expression of granzyme B was found in both the CD45RA⁺/effector and CD45RA⁻/memory compartments of TNF-α⁺/CD4⁺ and TNF-α⁺/CD8⁺ T-cells, further confirming the presence of circulating antigen experienced cytotoxic CD4⁺ T cells in the PB of hCMV-seropositive individuals (15).

Previous studies have reported that both PB memory CD4⁺ and CD8⁺ T cells from patients with persistent viral infections are phenotypically heterogeneous (29-31). Accordingly, EBV-specific and HIV-specific memory CD4⁺ T cells have been shown to display a predominance of CD45RA⁻/memory cells and an early central memory phenotype (CD45RA⁻/CCR7⁺) respectively, requiring targeting lymph nodes during reinfection in both conditions (29, 30). In contrast, our results indicate that hCMV memory CD4⁺ T cells show a functional profile consistent with an end-stage differentiated antigen-experienced phenotype with functional targets out of the lymph nodes (32). Similarly, we confirm that hCMV-specific CD8⁺ PB T cells from seropositive volunteers are equally distributed between the CD45RA⁻/memory and CD45RA⁺/effector compartments, with low/null CCR7 expression but intracytoplasmic expression of granzyme B (31). These findings would suggest that in subsequent reinfections, hCMV-specific CD8⁺ PB T cells may exert their cytotoxic effector function through the body without entering the lymph nodes.

Previous studies have shown the occurrence of oligoclonal expansions of CMV-specific T cells in seropositive individuals (20, 21). In the present study we confirm and extend such observations by showing that activated hCMV-specific CD4⁺ and CD8⁺ T-cells include a relatively restricted number of TCR-Vβ family members. Interestingly, predominance of some of the TCR-Vβ family members analyzed was noted. Furthermore, all except one of the donors showing anti-hCMV specific CD8⁺ T cells, showed expansions of TCR-Vβ family members common to those of the hCMV-specific CD4⁺ T lymphocytes. These results would support the notion that CD4⁺ T cells help to keep functionally robust memory CD8⁺ T cells (33, 34). Interestingly, although this helper role of CD4⁺ T cells is still under debate, it has been proposed that IL-2 also plays a role in the process (35, 36). Although at present there is no definitive evidence to classify IL-2 as a helper cytokine provided by CD4⁺ T cells for cross-primed CD8⁺ T cells, our results clearly show that activation of CMV-specific CD8⁺ T cells, but not CMV-specific CD4⁺ T cells, was associated with secretion of the cytokine. In turn, the expansion of common clonotypes in CD4⁺ and CD8⁺ PB T-cells from hCMV-seropositive individuals would also suggest the potential relevance of a group of CMV epitopes which are preferentially recognized by the expanded TCR-Vβ family members identified, as previously suggested by Bitmansour et al. (21).

Although the high number of hCMV proteins which are potentially immunogenic, only a few have been demonstrated to be capable of inducing immune responses in a relatively large number of individuals with different HLA haplotypes (37). Altogether, this would suggest that immunodominant epitopes need to be efficiently presented by specific HLA molecules. Accordingly, it has been suggested (38) that the stability of the antigen-HLA Class II molecules complex would have an important role in determining their specific immunogenicity. Interestingly, although the HLA haplotypes of the CMV-seropositive individuals were highly variable (37, 39, 40), in the present study we found a significant association between some HLA Class II and Class I haplotypes and the presence of specific TCR-Vβ clones of anti-hCMV CD4⁺ and CD8⁺ T-cells, respectively. Accordingly, expansion of TCR-Vβ13.1⁺ and TCR-Vβ16⁺ CD4⁺ T cells was associated with the HLA-DRB₁*0701, HLA-DQB₁*0202 and HLA-DQB₁*0402 haplotypes; in turn, expansion of TCR-Vβ4⁺ and TCR-Vβ5.1⁺ CD8⁺ T-cells was associated with the HLA-C*1203 haplotype. Altogether, these results would support the notion that the shape of the memory pool of hCMV-specific T-cells from these individuals is dictated by their own haplotypes.

Acknowledgements

This article is dedicated to all donors who helped the authors with this research. Thanks to Quentin Lécrevisse for his participation in the development of software tools for Flow Cytometric data analysis.

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Citing Literature

Volume72B, Issue5

September 2007

Pages 371-379

Association between the HLA haplotype and the TCR-Vβ repertoire of anti-hCMV specific memory T-cells in immunocompetent healthy adults