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Human virus-specific T cells in peripheral blood and lymph nodes: Phenotype,
function and clonal relationships
Remmerswaal, E.B.M.
Publication date
2014
Document Version
Final published version
Link to publication
Citation for published version (APA):
Remmerswaal, E. B. M. (2014). Human virus-specific T cells in peripheral blood and lymph
nodes: Phenotype, function and clonal relationships.
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DISCUSSION
9
GENERAL DISCUSSION
This thesis focusses on the characterization of T cell responses against (persistent) viral infections in peripheral blood and lymph nodes. We studied the immune response against the persistent viral infections human cytomegalovirus (hCMV), Epstein Barr virus (EBV) and human immunodeficiency virus (HIV) and compared these with the transient viral infections influenza A and respiratory syncytial virus (RSV).
Differences in virus genome and virion composition, genetic variation of the host (polymorphisms in genes encoding cytokines, HLA-type, expression of chemokine receptors etc), immunocompetence of the host, route and dose of infection and even infection history can all influence the immune response. In mice, all these factors can be standardized and therefore data from these experimental models do provide an invaluable insight in the mechanisms involved in the mammalian immune system. However, the goal of our studies was the characterization of such responses in man. Thus, although inter-individual and inter-viral differences will without doubt be large, we try to find common denominators of anti-viral T cell responses in man.
Because it is usually unknown when an individual becomes infected with a virus, most of the studies performed in humans are performed on T cells in the memory (for transient viral infections) or latent phase (for persistent viral infections). The majority of the data on primary viral infections are derived from murine models. However, the possibility that a naïve renal transplant recipient can receive a graft from a latently infected donor, allows for a unique opportunity to study primary viral infection in humans. The renal transplant setting also provided the possibility to study paired lymph node (LN) and PB samples. These LNs could often be found in the residual tissue that was removed during the anastomosis of the arteria and vena renalis when the iliac artery and vein were dissected free.
At least two parameters might influence the outcome of anti-viral responses in renal transplant patients when compared to healthy individuals. First, these patients are treated with an immunosuppressive drug regimen to prevent allograft rejection. Second, the route and dose of infection in renal transplant patients differs from the way healthy individuals become infected. In healthy individuals epithelial cells will become infected first with cell free virions. Renal transplant patients become infected by virions produced by latently infected cells within the graft once these cells have entered the lytic cycle. These patients can also become infected by cell to cell transfer of virus from graft cells that have entered the lytic cycle. However, in spite of these differences, these patients are usually able to control the viral replication without serious clinical complications and they do produce virus-specific IgM and IgG antibodies upon primary infection (1). Also, although anti-viral CD4+ and CD8+ T cell
responses in renal transplant recipients have shown to be larger, the quality of the response closely resembles that of healthy individuals (2, 3).
HCMV, EBV and HIV all persist (latently) after the primary infection. Although there are similarities between the methods these herpesviridae employ to escape the
DISCUSSION
9
immune system, there are also a lot of differences. Herpesviridae have co-evolved with their vertebrate hosts for more than 100 million years (4), which has allowed the generation of many immune escape mechanisms by the double-stranded, relatively stable DNA virus. In contrast, HIV-1 has probably been introduced from chimpanzees into the human species some 100 years ago (5, 6). Because of the highly error-prone reverse transcription, the single stranded positive-sense RNA viruses changes upon every cycle and adaptations to its environment occur much faster than will ever be possible in the herpesviridae. Also the tropism of EBV, hCMV and HIV is completely different, which will influence the immune system.
Phenotype of latent virus-specific CD4
+and CD8
+T cells
HCMV
The long co-evolution of the herpesviridae with its host has also allowed for many adaptations of the host to the virus. In spite of, or perhaps due to, the HLA-I downregulation, NK evasion and other immune evasive mechanisms, up to half of the total CD8+ T cells in the peripheral blood (PB) of individuals who are latently infected
with hCMV, can be dedicated to hCMV (chapter 5). In fact both cytotoxic CD8+ and
CD4+ T cells, expressing granzyme B and perforin, can be found in significantly higher
proportions in individuals latently infected with hCMV ((7, 8) and chapters 3 and 7). The phenotype of the majority of the latent phase PB hCMV-specific CD4+ and
CD8+ T cells is very distinctive. These effector-type cells contain vast amounts of the
cytolytic molecules granzyme B and perforin and display direct ex vivo cytotoxicity and have lost the co-stimulatory markers CD27 and CD28. We have shown that hCMV reactivation leads to higher numbers of hCMV-specific CD8+ T cells and
CD27 downregulation (9). The CD27 downregulation may be caused by ligation to its ligand CD70, which can be expressed on activated T cells, B cells and dendritic cells. Expression of CD70 on activated T cells was shown to be dependent on the cytokine milieu. Where IL-2 and IL-15 enhanced CD70 upregulation, IL-21 was shown to prevent it (9). Thus, the cytokine environment in response to a viral infection and the repetitive reactivations of a persistent infection can influence the CD27-expression on a responding T cell. Other support for this theory can be found when analyzing hCMV-specific CD8+ T cells in untreated HIV infected individuals. The hCMV-pp65
specific CD8+ T cells in HIV infected individuals express less CD27 than the
hCMV-pp65-specific CD8+ T cells in uninfected individuals.
HCMV can infect a large variety of different cell types like endothelial cells, epithelial cells and leukocytes. Since latency has been shown to occur in monocytes and CD34+
myeloid precursors, and reactivation has been shown to occur upon differentiation, hCMV infected monocyte-derived DCs can be expected to influence the immune response.
Upon T cell activation CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) can be upregulated. CD28 and CTLA4 compete for the same ligands CD80 (B7-1) and CD86
DISCUSSION
9
(B7-2) on antigen presenting cells. Ligation of CTLA4 leads to downregulation of the response and possibly tolerance by interference with TCR signals and the release of IDO from DCs (10-14). CTLA-4 has also been shown to enhance internalization and degradation of CD28 (15).Ligation of CD28 however leads to enhanced IL-2 production, proliferation and prevention of apoptosis (16, 17).
Latent phase PB hCMV-specific CD4+ and CD8+ T cells also express the chemokine
receptor CX3CR1 (12, 18), which allows them to migrate to fraktalkine (CX3CL1) expressing stressed (infected) endothelium and epithelium cells. Since hCMV-infected cells can also express vCX3CR1 (US28) (19, 20), the hCMV-specific T cells
might employ their CX3CR1 mediated migration to effectively cluster at places towards which infected leukocytes home.
EBV
As shown in chapter 2 and 3 the phenotype of EBNA3a-specific CD8+ T cells differs
from the BZLF1- and BMLF1-specific CD8+ T cells. Resting latent epitope-specific
CD8+ T cells usually still express both CD27 and CD28 and they contain granzyme
K. In contrast, half of the resting lytic cycle-specific CD8+ T cells have lost their CD28
expression and although a large proportion also contains granzyme K, a substantial amount does contain granzyme B.
No data have been presented about inhibition of EBV replication by granzyme K so far. However granzyme K has been shown to inhibit influenza virus replication via the disruption of the importin a1/b dimer, which is required for the transportation of viral NP to the nucleus (21). It has also been shown, just like all other granzymes, to target hnRNP K (heterogeneous nuclear ribonucleoprotein K), a multifunctional DNA/ RNA-binding protein involved in transcription, translation, splicing and mRNAstability. Granzyme M was shown to decrease hCMV-IE2 transcription and cell viability by cleavage of hnRNP K (22). It seems very likely that EBV-replication will be inhibited by similar mechanisms and because of the high percentage of EBV-specific CD8+ T cells
expressing granzyme K, this mechanism is most likely crucial in maintaining latency. In contrast to hCMV the tropism of EBV is rather limited. EBV infects epithelial cells and B cells, and latency can be established in memory B cells. Perhaps the lesser differentiated phenotype of EBV-specific CD8+ T cells can be explained by the fact
that EBV does not infect or affect professional APCs.
HIV
The main targets of HIV are CD4-expressing cells like T cells, dendritic cells, macrophages and monocytes. Although DCs are rather resistant to infection, depending on their C-type lectin expression they can affect the outcome of an HIV infection. DC-sign-expressing DCs can, by uptake of viral particles, infect CD4+ T cells
DISCUSSION
9
in Birbeck granules, if it was captured by langerin on Langerhans cells, thereby preventing further viral spread.
The individuals we studied in chapter 3 were untreated but had not yet progressed to AIDS. The loss of IL-7R
α
-expression on these cells probably reflects the very frequent engagement of the TCR, since we have shown that such an engagement causes prolonged downregulation of the IL-7Rα
(23). Downregulation due to binding of IL-7 to the IL-7R was shown to be transient.Temporal context of hCMV and EBV proteins
and CD8
+T cell responses
The above mentioned phenotype holds true for the latent phase CD8+ T cells against both
hCMV proteins studied: pp65 and IE1. This is quite remarkable since both proteins have different functions, different temporal contexts and they even differ in their presentation in HLA-I. Tegument protein pp65 is present in the virion and can therefore be processed and presented from the moment of infection. The transactivator IE1 however is the first protein to be produced after infection of a cell as the onset of the lytic cycle, but the already present pp65 can prevent presentation of IE1 derived peptides in HLA-I by phosphorylation of the IE1 protein. The balance between the amount of pp65 and IE1 is paramount in this blockade. With higher levels of another tegument protein, pp71, in the virion, IE transcription is enhanced and IE1 derived peptides are found in HLA-I (24). Indeed we have seen that the hCMV-pp65-specific CD8+ T cells often precede the
hCMV-IE1-specific CD8+ T cells (chapter 3 and data not shown) in primary hCMV.
In latently infected cells the IE1 protein marks the onset of hCMV reactivation. The tegument pp65 is a late protein and as such will be produced late during the viral cycle. Prevention of presentation of IE1 derived peptides in HLA-I can therefore not be prevented from the beginning of the reactivation. It has been shown in mice that reactivation of mCMV in lungs occurs frequently in a patchwork-like way (25, 26). It has also been shown that IE1-derived peptides indeed were presented in HLA-I and that IE1-specific CD8+ T cells terminated the mCMV reactivation (27). Thus, the dynamics
of pp65 and IE-derived peptide presentation in HLA-I and the subsequent pp65 and IE-specific CD8+ T cells are different during primary hCMV and reactivation and should
be considered when studying hCMV-specific CD8+ T cells.
It is also crucial to separate the different epitopes the response is directed to, when analyzing the EBV-specific CD8+ T cells. Both the IE and the EBNA proteins are not
present in the virion. The IE protein transactivator BZLF1 and the E protein BMLF1 are produced early in the lytic cycle and will therefore be produced by infected epithelial cells early in the primary infection. The EBNA3 proteins however are only transcribed in some of the latently infected B cells. When studying primary EBV infections we did indeed observe that the lytic-epitope (BZLF1 and BMLF1) specific CD8+ T cells always
preceded the PB EBNA-specific CD8+ T cells (chapter 3 and 6).
DISCUSSION
9
Clonal breadth of virus-specific CD8
+T cells
CD8+ T cell clones directed towards hCMV-IE- and pp65-derived epitopes are among
the largest clones in the CD8 compartment (chapter 3). IE-specific and pp65-specific CD8+ T cells however do differ significantly in their clonal breadth. In chapter 3 we show
that hCMV-pp65-specific CD8+ T cells generally consist of substantially more clones than
hCMV-IE specific CD8+ T cells. Exceptions to this rule do exist (chapter 3 and chapter 6) as
we have seen healthy individuals and renal transplant patients who mounted a monoclonal response against an hCMV-pp65 derived peptide and we have found one renal transplant patient who elicited a relatively broad response against an hCMV-IE epitope.
Indeed when hCMV-IE specific CD8+ T cells appear only late during the primary
hCMV, the cytokine milieu, availability of nutrients, co-stimulation, abundance of cognate peptide amongst others will differ considerably. For example, the pro-apoptotic protein Noxa has been shown to be upregulated at times of nutrient and cytokine deprivation and the absence of Noxa has been shown to result in enhanced diversity of the effector pool (28). Thus one might envision that during the onset of the immune response, when all nutrients and cytokines are abundant, Noxa will be low and lower affinity clones can expand too. In contrast at the height of the immune response competition for nutrients and cytokines will occur; Noxa will be upregulated and only higher affinity clones will expand. Thus, the early pp65-specific clones might be more diverse in their affinity and their clonal repertoire than the IE-specific clones. And when the hCMV-IE response is broad, it could be the result of an early hCMV-IE response, possibly caused by a pp71 favored balance of tegument proteins. Another reason for the difference in clonal breadth might be the fact that latently infected monocytes can differentiate into dendritic cells. Differentiation will lead to the onset of viral replication and the hCMV immune escape mechanism within these DCs are likely to influence both the clonal breadth and the resulting phenotype.
The difference in clonal breadth that was observed in the CD8 response towards hCMV-proteins is similar to that of EBNA-specific clones when compared to the BZLF-specific clones. CD8+ T cell responses towards EBNA3a and b derived peptides tend
to consist of fewer clones than those directed towards BZLF1 derived peptides. Once again, this might be related to the elapsed time after the onset of viral replication, but also on the cell type presenting the antigens.
Clonal evolution of CD8
+T cell responses
Primary infection
We show in chapter 3 that the clonal repertoire of both CD8+ T cells directed against
EBV-lytic and latent epitopes as well as hCMV-IE and pp65-specific CD8+ T cells remains
rather stable for over more than 3 years after the primary infection. Others have shown that the acute phase response focusses early during the primary infection, leaving a more restricted repertoire in latency (29). For this last study, samples as early as two weeks after
DISCUSSION
9
infection were analyzed, whereas in chapter 3 we analyzed the samples once the tetramer positive population was big enough to detect the individual clones within the total CD8+
population. Thus, we might have missed this early focussing. On the other hand, when frequencies are so very low, the risk of impurities in the sorts are also more substantial and in the days before next generation sequencing, these impurities were harder to exclude in the analyses, due to the many separate PCR’s that had to be performed.
In chapter 6 we studied the primary hCMV-pp65 response in two additional patients. This time, we did split the IL-7R
α
-expressing and negative hCMV-specific CD8+ T cells. In mice it was shown that the IL-7Rα
-expressing population containedthe long-lived precursor cells. We sorted the first possible time point at which sufficient IL-7R
α
-expressing cells were present. These time points were earlier in the response than the time points taken in chapter 3 and the patients were different.In chapter 6 we showed that IL-7R
α
expressing hCMV-pp65-specific CD8+ T cellsfrom the acute phase of the response are not the precursors for the latent phase T cells. Intuitively this seems logical. Circulating hCMV-specific cells would be exposed to their antigen with every reactivation that results in viral replication, leading to depletion or exhaustion of the precursor pool. The buffered memory theory, suggesting a location where such a precursor pool would be hidden from antigenic challenges unless the antigenic burden was large enough to reach such a site offers a reasonable theory (30).
Reactivation / reinfection
Endogenous reactivation of hCMV can occur because of the immunosuppressive treatment of the renal transplant patient, which requires adjustments of the immune response to maintain control of virus. However, when the donor also is latently infected with hCMV, infection with the donor derived virus can occur too.
As explained above, the IE1 protein is the first to be produced upon viral reactivation and generally these reactivations is effectively controlled by T cells before the actual production of new virions. However, such reactivations will be hard to study, since one can not detect a viral load. We have studied renal transplant recipients who were latently infected before transplantation and in whom we could measure hCMV-DNA in the peripheral blood compartment (chapter 6).
In cases where the donor was latently infected with hCMV too, reactivation of hCMV in donor derived cells would require the recognition of the HLA-epitope combination on the donor derived cells. It will therefore depend on the matching between recipient and donor HLA and the HLA-peptide combinations against which the recipient has mounted a response. If the recipient’s hCMV-IE-specific CD8+ T cells can not recognize the
reactivating donor derived cells, they will be unable to eliminate them before the release of new virions. In such cases, the interplay between pp65 and pp71 with IE1 in the newly infected recipients cells, will once again determine the time of response of CD8+ T cells.
This might account for the lack of expansion of IE1-specific CD8+ T cell responses at the
DISCUSSION
9
moment of hCMV replication when compared to the pp65-directed responses. However, the IE1-specific CD8+ T cells in these patients can respond to suppress the endogenous
reactivation of their own cells even before viral replication occurs.
We showed that upon viral reactivation or reinfection, apparently new clones can be recruited into the PB hCMV-IE- and pp65-specific CD8+ T cell pool, depending
on the breadth of the pre-transplantation pool. If a hCMV-specific response was highly restricted before reactivation it remained monoclonal, suggesting that these individuals either mounted a very adequate response during primary infection or that the T cells directed against this epitope are actually not involved (epitope is not (yet) presented, or T cells are exhausted). We have shown that when hCMV-specific CD8+ T cells are stimulated with their cognate antigen in the optimal milieu, they
can proliferate, produce cytokines and effectively kill the loaded target (31). Thus, exhaustion of these cells seems an unlikely option.
The fact that the monoclonal pp65-specific CD8+ T cells did become activated and
expanded in the PB suggests that these cells did see their antigen. That leaves the possibility of the optimal monoclonal response during primary infection, implying that a broad repertoire might not be optimal. The recruitment of new clones as a result of the hCMV-reactivation in patients who had an oligoclonal CD8+ T cell response
before reactivation also suggest that the pre transplantation clones were not optimal. However, many healthy donors do have oligoclonal CD8+ T cell responses, and they
do not experience major adverse events of the hCMV infection.
It has been shown that a higher percentage of the CD45RA re-expressing effector-type hCMV-pp65-specific CD8+ T cells when compared to their CD45R0-expressing
counterparts had a lower antigen binding affinity (32). This was especially seen in elderly individuals and depended on the size of the hCMV-specific CD8+ T cell pool
(32). It was also shown that this CD45RA re-expression could occur (at least in vitro) by exposure to IL-15 possibly produced by monocytes (32).
We have shown that hCMV reactivation caused both downregulation of CD27 and expansion of the pre-existing pool of hCMV-pp65-specific CD8+ T cells (9). Moreover
the size of the effector-type cell pool within the total hCMV-pp65 CD8+ T cells has
been shown to correlate with the total size of this pool (9). Thus it seems likely that upon every reactivation either more lower antigen binding affinity cells are recruited into the PB hCMV-pp65 specific CD8+ T cell pool, or the lower affinity hCMV-specific
CD8+ T cells are expanding or surviving relatively more than the higher affinity
hCMV-pp65-specific CD8+ T cells.
The first option would confirm the recruitment of new clones into the PB especially in individuals who already have a broader clonal repertoire. The fact that we sometimes see some small new clones appearing in individuals with a monoclonal response before reactivation might be the onset of such a broader repertoire.
The second option would suggest that the high affinity clones would be lost over time, either due to the fact that get outgrown or because they die after being activated.
DISCUSSION
9
The affinity data need to be confirmed for other hCMV-specific CD8+ T cells like
IE1-specific CD8+ T cells.
Virus-specific CD8
+T cells in lymph nodes
Where virus-specific CD8+ T cells in PB can be strikingly different depending on the virus
or even the protein against which they have been generated, hCMV-, EBV and influenza A-specific CD8+ T cells in LNs actually quite resemble each other in number, phenotype
and function (chapter 5). We have also shown that LNs actually contained unique hCMV-, EBV and influenza A-specific CD8+ T clones that could not be found in the PB.
LN are required for the priming of naïve T cells, but lesser so during memory responses and reactivations as antigen experienced T cells do not need professional APCs to become activated. These data might suggest that virus-specific CD8+ T
cells in LN either form a reservoir for memory cells, shielded from constant antigenic stimulation, or that they have a special function within the lymph node. In chapter 7 we showed that the LN probably may not function as such a memory cell reservoir, which suggests that these cells indeed are tissue resident T cells, with a possible local function. One such function could be the elimination of dendritic cells that present epitopes that are already efficiently recognized by the existing pool of antigen experienced cells. Regardless of the phenotype of a resting antigen-experienced cell one can expect that upon reengagement of the T cell with its antigen, cytotoxic molecules, like granzyme B and perforin, will be upregulated, allowing them to effectively kill the antigen expressing cell. It has been suggested before that homing of CXCR3-expressing T cells to draining lymph nodes could indeed abrogate the ongoing immune response after a primary infection by eliminating the antigen presenting DCs (33). Thus, it seems possible that these cells remain and prevent a second round of activation of naive T cells upon reactivation.
Of course the question remains if a memory cell pool shielded from the PB does exist and if so, where this buffered memory pool would be located. Based on previous publications, two locations could harbor such a pool. The first one is the spleen and the second the bone marrow. HCMV-specific CD8+ T cells have been found in both
tissues (34-36). However, the phenotype and functions of the hCMV-specific CD8+ T
cells in these tissues still need thorough investigation. In mice it was shown that CCR7-expressing memory cells were able to home to LN and the white pulpa of the spleen, whereas CCR7-negative cells occupied the red pulpa (37, 38). An interesting feature of the hCMV-specific CD8+ T cells in the bone marrow is the possible presence of the
latently infected CD34+ myeloid precursors which might actually reside there. Further
research on the phenotype, function and the clonal relationships of the hCMV and EBV-specific CD8+ T cells compared to their PB counterparts will give further insight
into the sustenance of immune responses against persistent viral infections.
DISCUSSION
9
Cytotoxic hCMV-specific CD4
+T cells
Recently it has been shown that cytotoxic CD4+ T cells differ from the CD4+ T helper
cells with respect to the expression of the T helper (Th) lineage master regulator, the Th-inducing BTB/POZ domain-containing Kruppel-like zinc-finger transcription factor, ThPOK (39). In CD8+ T cells ThPOK expression is repressed in the thymus by the
expression of RUNX3 (runt-related transcription factor 3). In cytotoxic CD4+ T cells
however the loss of ThPOK expression occurs post thymically as a result of repeated stimulation with their cognate antigen in vivo (39). RUNX3 is upregulated in activated CD4+ T cells by T-Bet and promotes the Th1 program and subsequent IFN
γ
productionby CD4+ T cells. The loss of ThPOK expression leads to the de-repression of the
cytolytic-gene expression program, which causes the cytolytic protein expression (40). Other indications that cytotoxic hCMV-specific CD4+ T cells emerge as a result
of repetitive antigenic stimulation are the loss of CD27 and the fact that we observe significantly higher percentages of these cells in immunocompromised patients when compared to healthy donors.
In chapter 7 and 8 we showed that CD28
⁻
CD4+ T cells are hCMV-specific CD4+T cells (chapter 7 and 8). They could only be found in individuals latently infected with hCMV, appeared after primary hCMV infection, produced IFN
γ
and proliferated after stimulation with antigen, and displayed ex vivo cytotoxicity against hCMV-antigen or pp65-derived peptide-loaded target cells. Phenotypically and functionally they resembled their hCMV-specific CD8+ T cell counterparts. They containedgranzyme B and perforin, lost their expression of CD27 and CD28, and do express CX3CR1 (18). They also produced IFN
γ
, TNFα
, MIP-1β
, but no IL-2 and expressed CD107a after stimulation with their cognate peptide (41). Lastly, the cytotoxic CD4+T cells also had a very limited clonal repertoire, which overlapped minimally with the acute phase IFN
γ
-producing CD4+ T cells (chapter 8).We have shown that at least part of these cells recognize a pp65 derived epitope. It has also been shown that gB (Glycoprotein B, involved in cell entry)-specific CD4+
T cells display direct ex-vivo cytotoxicity towards endogenous gB but not exogenous gB presented by epithelial cells, endothelial cells and glial cells via autophagy (42). GB-specific CD4+ T cells can lyse lytically infected monocytes, but not latently infected
monocytes (43). Also HCMV-IE specific CD4+ T cells have been shown to produce IFN
γ
.We tested the specificity of the CD28
⁻
CD4+ T cell by stimulation with hCMV-Ag,and showed that not all cells proliferated or produced antigen. A drawback of the use of antigen is that it will enter the exogenous presentation route, and since it was shown that only endogenous gB was recognized by gB-specific CD4+ T cells, the
gB-specific CD4+ T cells will not recognize the hCMV-antigen loaded target cells and thus
not proliferate, produce cytokine and lyse the loaded target cell. Responses against other hCMV-proteins might be affected in a similar fashion.
In addition, the hCMV-antigen we used is derived from the MRC-5 cells infected with the hCMV AD169 strain (Microbix Biosystems Inc). This strain has been shown to have
DISCUSSION
9
a 1.5kb deletion in its genome (UL133-151), when compared to the clinical strains. The deleted genes encode proteins that are involved in immune evasion, viral chemokines and proteins that determine cell type specific outcome of infection. This implies that at least part of the CD28
⁻
CD4+ T cells might be directed against peptides derived from one ormore of these, in AD169 absent, proteins. Indeed it has been shown that UL138 (a protein that mediates suppression of viral replication (43))-specific CD4+ T cells do exist in healthy
hCMV seropositive subjects (44). These UL138-specific CD4+ T cells were shown to indeed
display HLA-II restricted cytotoxicity and produced IFN
γ
upon stimulation.Thus, some of the acute phase lytic epitope (like pp65, gB and IE)-specific hCMV-specific CD28+CD4+ T clones and the somewhat later appearing latent epitope (like
UL138) specific hCMV-specific CD4+ T cell clones might give rise to the cytotoxic
CD28
⁻
CD4+ T cells upon repetitive exposure to their cognate antigen.OVERALL CONCLUSION
The combination of multifluorochrome flowcytometry and next generation sequencing used in this thesis shows that it is possible to characterize many aspects of the human anti-viral immune response.
Thus, this thesis paves the way for a better understanding of the phenotype, function and clonal relationships of virus-specific CD4+ and CD8+ T cells directed against many more
proteins derived from many more viruses in a wide variety of tissues. The fact that an ever smaller sample can be used to perform such an analysis without loss of information, will allow for the study of virus-specific CD4+ and CD8+ T cells with extremely low frequencies
and the analysis of anti-viral responses in small samples such as biopsies.
DISCUSSION
9
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