The Absence of M1 Leads to Increased Establishment of Murine Gammaherpesvirus 68 Latency in IgD-Negative B Cells

The secreted M1 protein of murine gammaherpesvirus 68 (MHV68) promotes effector Vβ4⁺ CD8⁺ T cell expansion to impact virus control and immune-mediated pathologies in C57BL/6 mice, but not BALB/c mice. We report a striking increase in the number of genome-positive, IgD⁻ B cells during chronic infection of both mouse strains. This suggests a novel role for M1 in influencing long-term maintenance in a major latency reservoir irrespective of the degree of Vβ4⁺ CD8⁺ T cell expansion.     

High Affinity of Interaction between Superantigen and T Cell Receptor Vβ Molecules Induces a High Level and Prolonged Expansion of Superantigen-reactive CD4+ T Cells

In mice implanted with an osmotic pump filled with the superantigen (SAG) staphylococcal enterotoxin A (SEA), the Vβ3⁺CD4⁺ T cells exhibited a high level of expansion whereas the Vβ11⁺CD4⁺ T cells exhibited a mild level of expansion. In contrast, in mice implanted with an osmotic pump filled with SE-like type P (SElP, 78.1% homologous with SEA), the Vβ11⁺CD4⁺ T cells exhibited a high level of expansion while the Vβ3⁺CD4⁺ T cells exhibited a low level of expansion, suggesting that the level of the SAG-induced response is determined by the affinities between the TCR Vβ molecules and SAG. Analyses using several hybrids of SEA and SElP showed that residue 206 of SEA determines the response levels of Vβ3⁺CD4⁺ and Vβ11⁺CD4⁺ T cells both in vitro and in vivo. Analyses using the above-mentioned hybrids showed that the binding affinities between SEA and the Vβ3/Vβ11 β chains and between SEA-MHC class II-molecule complex and Vβ3⁺/Vβ11⁺ CD4⁺ T cells determines the response levels of the SAG-reactive T cells both in vitro and in vivo.     

Increase in IFNγ?IL-2+ Cells in Recent Human CD4 T Cell Responses to 2009 Pandemic H1N1 Influenza

Human CD4 T cell recall responses to influenza virus are strongly biased towards Type 1 cytokines, producing IFNγ, IL-2 and TNFα. We have now examined the effector phenotypes of CD4 T cells in more detail, particularly focusing on differences between recent versus long-term, multiply-boosted responses. Peptides spanning the proteome of temporally distinct influenza viruses were distributed into pools enriched for cross-reactivity to different influenza strains, and used to stimulate antigen-specific CD4 T cells representing recent or long-term memory. In the general population, peptides unique to the long-circulating influenza A/New Caledonia/20/99 (H1N1) induced Th1-like responses biased toward the expression of IFNγ⁺TNFα⁺ CD4 T cells. In contrast, peptide pools enriched for non-cross-reactive peptides of the pandemic influenza A/California/04/09 (H1N1) induced more IFNγ⁻IL-2⁺TNFα⁺ T cells, similar to the IFNγ⁻IL-2⁺ non-polarized, primed precursor T cells (Thpp) that are a predominant response to protein vaccination. These results were confirmed in a second study that compared samples taken before the 2009 pandemic to samples taken one month after PCR-confirmed A/California/04/09 infection. There were striking increases in influenza-specific TNFα⁺, IFNγ⁺, and IL-2⁺ cells in the post-infection samples. Importantly, peptides enriched for non-cross-reactive A/California/04/09 specificities induced a higher proportion of Thpp-like IFNγ⁻IL-2⁺TNFα⁺ CD4 T cells than peptide pools cross-reactive with previous influenza strains, which induced more Th1 (IFNγ⁺TNFα⁺) responses. These IFNγ⁻IL-2⁺TNFα⁺ CD4 T cells may be an important target population for vaccination regimens, as these cells are induced upon infection, may have high proliferative potential, and may play a role in providing future effector cells during subsequent infections.     

Clonal Expansions of CD8+ T Cells with IL-10 Secreting Capacity Occur during Chronic Mycobacterium tuberculosis Infection

The exact role of CD8⁺ T cells during Mycobacterium tuberculosis (Mtb) infection has been heavily debated, yet it is generally accepted that CD8⁺ T cells contribute to protection against Mtb. In this study, however, we show that the Mtb-susceptible CBA/J mouse strain accumulates large numbers of CD8⁺ T cells in the lung as infection progresses, and that these cells display a dysfunctional and immunosuppressive phenotype (PD-1⁺, Tim-3⁺, CD122⁺). CD8⁺ T cell expansions from the lungs of Mtb-infected CBA/J mice were also capable of secreting the immunosuppressive cytokine interleukin-10 (IL-10), although in vivo CD8⁺ T cell depletion did not significantly alter Mtb burden. Further analysis revealed that pulmonary CD8⁺ T cells from Mtb-infected CBA/J mice were clonally expanded, preferentially expressing T cell receptor (TcR) Vβ chain 8 (8.2, 8.3) or Vβ 14. Although Vβ8⁺ CD8⁺ T cells were responsible for the majority of IL-10 production, in vivo depletion of Vβ8⁺ did not significantly change the outcome of Mtb infection, which we hypothesize was a consequence of their dual IL-10/IFN-γ secreting profiles. Our data demonstrate that IL-10-secreting CD8⁺ T cells can arise during chronic Mtb infection, although the significance of this T cell population in tuberculosis pathogenesis remains unclear.     

A Human Cell Line Model for Interferon-α Driven Dendritic Cell Differentiation

The CD34⁺ MUTZ-3 acute myeloid leukemia cell line has been used as a dendritic cell (DC) differentiation model. This cell line can be cultured into Langerhans cell (LC) or interstitial DC-like cells using the same cytokine cocktails used for the differentiation of their primary counterparts. Currently, there is an increasing interest in the study and clinical application of DC generated in the presence of IFNα, as these IFNα-DC produce high levels of inflammatory cytokines and have been suggested to be more potent in their ability to cross-present protein antigens, as compared to the more commonly used IL-4-DC. Here, we report on the generation of IFNα-induced MUTZ-DC. We show that IFNα MUTZ-DC morphologically and phenotypically display characteristic DC features and are functionally equivalent to “classic” IL-4 MUTZ-DC. IFNα MUTZ-DC ingest exogenous antigens and can subsequently cross-present HLA class-I restricted epitopes to specific CD8⁺ T cells. Importantly, mature IFNα MUTZ-DC express CCR7, migrate in response to CCL21, and are capable of priming naïve antigen-specific CD8⁺ T cells. In conclusion, we show that the MUTZ-3 cell line offers a viable and sustainable model system to study IFNα driven DC development and functionality.     

The Contribution of Non-Conventional T Cells and NK Cells in the Mycobacterial-Specific IFNγ Response in Bacille Calmette-Guérin (BCG)-Immunized Infants

Background

The Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccine is given to >120 million infants each year worldwide. Most studies investigating the immune response to BCG have focused on adaptive immunity. However the importance of TCR-gamma/delta (γδ) T cells and NK cells in the mycobacterial-specific immune response is of increasing interest.

Methods

Participants in four age-groups were BCG-immunized. Ten weeks later, in vitro BCG-stimulated blood was analyzed for NK and T cell markers, and intracellular IFNgamma (IFNγ) by flow cytometry. Total functional IFNγ response was calculated using integrated median fluorescence intensity (iMFI).

Results

In infants and children, CD4 and CD4-CD8- (double-negative (DN)) T cells were the main IFNγ-expressing cells representing 43-56% and 27-37% of total CD3+ IFNγ+ T cells respectively. The iMFI was higher in DN T cells compared to CD4 T cells in all age groups, with the greatest differences seen in infants immunized at birth (p=0.002) or 2 months of age (p<0.0001). When NK cells were included in the analysis, they accounted for the majority of total IFNγ-expressing cells and, together with DN Vδ2 γδ T cells, had the highest iMFI in infants immunized at birth or 2 months of age.

Conclusion

In addition to CD4 T cells, NK cells and DN T cells, including Vδ2 γδ T cells, are the key populations producing IFNγ in response to BCG immunization in infants and children. This suggests that innate immunity and unconventional T cells play a greater role in the mycobacterial immune response than previously recognized and should be considered in the design and assessment of novel tuberculosis vaccines.     

Primary clearance of murine gammaherpesvirus 68 by PKCtheta-/- CD8 T cells is compromised in the absence of help from CD4 T cells

CD4 T cells are dispensable for acute control of murine gammaherpesvirus 68 (MHV-68) but are necessary for effective long-term control of the virus by CD8 T cells. In contrast, protein kinase C θ (PKCθ) is not essential for either acute or long-term viral control. However, we found that while either CD4 or CD8 T cells could mediate the clearance of MHV-68 from the lungs of PKCθ^+/+ mice, PKCθ^−/− mice depleted of either subset failed to clear the virus. These data suggest that there are two alternative pathways for MHV-68 clearance, one dependent on CD4 T cells and the other on PKCθ. Protection mediated by the latter appears to be short-lived. These observations may help to explain the differential requirement for PKCθ in various models of CD8 T-cell activation and differences in the costimulatory requirements for acute and long-term viral control.     

T-cell receptor Vβ repertoire of CD8+ T-lymphocyte subpopulations in cutaneous leishmaniasis patients from the state of Rio de Janeiro, Brazil

In human cutaneous leishmaniasis (CL), the immune response is mainly mediated by T-cells. The role of CD8⁺ T-lymphocytes, which are related to healing or deleterious functions, in affecting clinical outcome is controversial. The aim of this study was to evaluate T-cell receptor diversity in late-differentiated effector (LDE) and memory CD8⁺ T-cell subsets in order to create a profile of specific clones engaged in deleterious or protective CL immune responses. Healthy subjects, patients with active disease (PAD) and clinically cured patients were enrolled in the study. Total CD8⁺ T-lymphocytes showed a disturbance in the expression of the Vβ2, Vβ9, Vβ13.2, Vβ18 and Vβ23 families. The analyses of CD8⁺T-lymphocyte subsets showed high frequencies of LDE CD8⁺T-lymphocytes expressing Vβ12 and Vβ22 in PAD, as well as effector-memory CD8⁺ T-cells expressing Vβ22. We also observed low frequencies of effector and central-memory CD8⁺ T-cells expressing Vβ2 in PAD, which correlated with a greater lesion size. Particular Vβ expansions point to CD8⁺ T-cell clones that are selected during CL immune responses, suggesting that CD8⁺ T-lymphocytes expressing Vβ12 or Vβ22 are involved in a LDE response and that Vβ2 contractions in memory CD8⁺T-cells are associated with larger lesions.     

HDAC1 Controls CD8+ T Cell Homeostasis and Antiviral Response

Reversible lysine acetylation plays an important role in the regulation of T cell responses. HDAC1 has been shown to control peripheral T helper cells, however the role of HDAC1 in CD8⁺ T cell function remains elusive. By using conditional gene targeting approaches, we show that LckCre-mediated deletion of HDAC1 led to reduced numbers of thymocytes as well as peripheral T cells, and to an increased fraction of CD8⁺CD4^– cells within the CD3/TCRβ^lo population, indicating that HDAC1 is essential for the efficient progression of immature CD8⁺CD4^– cells to the DP stage. Moreover, CD44^hi effector CD8⁺ T cells were enhanced in mice with a T cell-specific deletion of HDAC1 under homeostatic conditions and HDAC1-deficient CD44^hi CD8⁺ T cells produced more IFNγ upon ex vivo PMA/ionomycin stimulation in comparison to wild-type cells. Naïve (CD44^l°CD62L⁺) HDAC1-null CD8⁺ T cells displayed a normal proliferative response, produced similar amounts of IL-2 and TNFα, slightly enhanced amounts of IFNγ, and their in vivo cytotoxicity was normal in the absence of HDAC1. However, T cell-specific loss of HDAC1 led to a reduced anti-viral CD8⁺ T cell response upon LCMV infection and impaired expansion of virus-specific CD8⁺ T cells. Taken together, our data indicate that HDAC1 is required for the efficient generation of thymocytes and peripheral T cells, for proper CD8⁺ T cell homeostasis and for an efficient in vivo expansion and activation of CD8⁺ T cells in response to LCMV infection.     

High-Throughput Sequencing of Islet-Infiltrating Memory CD4+ T Cells Reveals a Similar Pattern of TCR Vβ Usage in Prediabetic and Diabetic NOD Mice

Autoreactive memory CD4⁺ T cells play a critical role in the development of type 1 diabetes, but it is not yet known how the clonotypic composition and TCRβ repertoire of the memory CD4⁺ T cell compartment changes during the transition from prediabetes to diabetes. In this study, we used high-throughput sequencing to analyze the TCRβ repertoire of sorted islet-infiltrating memory CD4⁺CD44^high T cells in 10-week-old prediabetic and recently diabetic NOD mice. We show that most clonotypes of islet-infiltrating CD4⁺CD44^high T cells were rare, but high-frequency clonotypes were significantly more common in diabetic than in prediabetic mice. Moreover, although the CD4⁺CD44^high TCRβ repertoires were highly diverse at both stages of disease development, dominant use of TRBV1 (Vβ2), TRBV13-3 (Vβ8.1), and TRBV19 (Vβ6) was evident in both prediabetic and diabetic mice. Our findings strongly suggest that therapeutic targeting of cells specifically expressing the dominant TCRβ might reduce pancreatic infiltration in prediabetic mice and attenuate the progression to diabetes.     

Phosphoantigen/IL2 Expansion and Differentiation of Vγ2Vδ2 T Cells Increase Resistance to Tuberculosis in Nonhuman Primates

Dominant Vγ2Vδ2 T-cell subset exist only in primates, and recognize phosphoantigen from selected pathogens including M. tuberculosis(Mtb). In vivo function of Vγ2Vδ2 T cells in tuberculosis remains unknown. We conducted mechanistic studies to determine whether earlier expansion/differentiation of Vγ2Vδ2 T cells during Mtb infection could increase immune resistance to tuberculosis in macaques. Phosphoantigen/IL-2 administration specifically induced major expansion and pulmonary trafficking/accumulation of phosphoantigen-specific Vγ2Vδ2 T cells, significantly reduced Mtb burdens and attenuated tuberculosis lesions in lung tissues compared to saline/BSA or IL-2 controls. Expanded Vγ2Vδ2 T cells differentiated into multifunctional effector subpopulations capable of producing anti-TB cytokines IFNγ, perforin and granulysin, and co-producing perforin/granulysin in lung tissue. Mechanistically, perforin/granulysin-producing Vγ2Vδ2 T cells limited intracellular Mtb growth, and macaque granulysin had Mtb-bactericidal effect, and inhibited intracellular Mtb in presence of perforin. Furthermore, phosphoantigen/IL2-expanded Vγ2Vδ2 T effector cells produced IL-12, and their expansion/differentiation led to enhanced pulmonary responses of peptide-specific CD4+/CD8+ Th1-like cells. These results provide first in vivo evidence implicating that early expansion/differentiation of Vγ2Vδ2 T effector cells during Mtb infection increases resistance to tuberculosis. Thus, data support a rationale for conducting further studies of the γδ T-cell-targeted treatment of established TB, which might ultimately help explore single or adjunctive phosphoantigen expansion of Vγ2Vδ2 T-cell subset as intervention of MDR-tuberculosis or HIV-related tuberculosis.     

Cytokine Diversity in the Th1-Dominated Human Anti-Influenza Response Caused by Variable Cytokine Expression by Th1 Cells,and a Minor Population of Uncommitted IL-2+IFNγ- Thpp Cells

Within overall Th1-like human memory T cell responses, individual T cells may express only some of the characteristic Th1 cytokines when reactivated. In the Th1-oriented memory response to influenza, we have tested the contributions of two potential mechanisms for this diversity: variable expression of cytokines by a uniform population during activation, or different stable subsets that consistently expressed subsets of the Th1 cytokine pattern. To test for short-term variability, in vitro-stimulated influenza-specific human memory CD4+ T cells were sorted according to IL-2 and IFNγ expression, cultured briefly in vitro, and cytokine patterns measured after restimulation. Cells that were initially IFNγ+ and either IL-2+ or IL-2- converged rapidly, containing similar proportions of IL-2-IFNγ+ and IL-2+IFNγ+ cells after culture and restimulation. Both phenotypes expressed Tbet, and similar patterns of mRNA. Thus variability of IL-2 expression in IFNγ+ cells appeared to be regulated more by short-term variability than by stable differentiated subsets. In contrast, heterogeneous expression of IFNγ in IL-2+ influenza-specific T cells appeared to be due partly to stable T cell subsets. After sorting, culture and restimulation, influenza-specific IL-2+IFNγ- and IL-2+IFNγ+ cells maintained significantly biased ratios of IFNγ+ and IFNγ- cells. IL-2+IFNγ- cells included both Tbet^lo and Tbet^hi cells, and showed more mRNA expression differences with either of the IFNγ+ populations. To test whether IL-2+IFNγ-Tbet^lo cells were Thpp cells (primed but uncommitted memory cells, predominant in responses to protein vaccines), influenza-specific IL-2+IFNγ- and IL-2+IFNγ+ T cells were sorted and cultured in Th1- or Th2-generating conditions. Both cell types yielded IFNγ-secreting cells in Th1 conditions, but only IL-2+IFNγ- cells were able to differentiate into IL-4-producing cells. Thus expression of IL-2 in the anti-influenza response may be regulated mainly by short term variability, whereas different T cell subsets, Th1 and Thpp, may contribute to variability in IFNγ expression.     

The Co-Stimulatory Effects of MyD88-Dependent Toll-Like Receptor Signaling on Activation of Murine γδ T Cells

γδ T cells express several different toll-like receptor (TLR)s. The role of MyD88- dependent TLR signaling in TCR activation of murine γδ T cells is incompletely defined. Here, we report that Pam3CSK4 (PAM, TLR2 agonist) and CL097 (TLR7 agonist), but not lipopolysaccharide (TLR4 agonist), increased CD69 expression and Th1-type cytokine production upon anti-CD3 stimulation of γδ T cells from young adult mice (6-to 10-week-old). However, these agonists alone did not induce γδ T cell activation. Additionally, we noted that neither PAM nor CL097 synergized with anti-CD3 in inducing CD69 expression on γδ T cells of aged mice (21-to 22-month-old). Compared to young γδ T cells, PAM and CL097 increased Th-1 type cytokine production with a lower magnitude from anti-CD3- stimulated, aged γδ T cells. Vγ1⁺ and Vγ4⁺ cells are two subpopulations of splenic γδ T cells. PAM had similar effects in anti-CD3-activated control and Vγ4⁺ subset- depleted γδ T cells; whereas CL097 induced more IFN-γ production from Vγ4⁺ subset-depleted γδ T cells than from the control group. Finally, we studied the role of MyD88-dependent TLRs in γδ T cell activation during West Nile virus (WNV) infection. γδ T cell, in particular, Vγ1⁺ subset expansion was significantly reduced in both MyD88- and TLR7- deficient mice. Treatment with TLR7 agonist induced more Vγ1⁺ cell expansion in wild-type mice during WNV infection. In summary, these results suggest that MyD88-dependent TLRs provide co-stimulatory signals during TCR activation of γδ T cells and these have differential effects on distinct subsets.     

Human CD8+ T Cells from TB Pleurisy Respond to Four Immunodominant Epitopes in Mtb CFP10 Restricted by HLA-B Alleles

CD8⁺ T cells are essential for host defense to Mycobacterium tuberculosis (Mtb) infection and identification of CD8⁺ T cell epitopes from Mtb is of importance for the development of effective peptide-based diagnostics and vaccines. We previously demonstrated that the secreted 10-KDa culture filtrate protein (CFP10) from Mtb is a potent CD8⁺ T cell antigen but the repertoire and dominance pattern of human CD8 epitopes for CFP10 remained poorly characterized. In the present study, we undertook to define immunodominant CD8 epitopes involved in CFP10 using a panel of CFP10-derived 13–15 amino acid (aa) peptides overlapping by 11 aa. Four peptides in CFP10 were observed to induce significant CD8⁺ T cell responses and we further determined the size of the epitopes involved in each individual peptide tested. Four 9 aa CD8 epitopes were finally identified and deleting a single amino acid from the N or C terminus of either peptide markedly reduced IFN-γ production, suggesting that they are minimum of CD8 epitopes. In the individuals tested, each epitope represented a single immunodominant response in CD8⁺ T cells. The epitope-specific CD8⁺ T cells displayed effector or effector memory phenotypes and could upregulate the expression of CD107a/b upon antigen stimulation. In addition, we found that epitope-specific CD8⁺ T cells shared biased usage of T cell receptor (TCR) variable region of β chain (Vβ) 12, 9, 7.2 or Vβ4 chains. As judged from HLA-typing results and using bioinformatics technology for prediction of MHC binding affinity, we found that the epitope-specific CD8⁺ T cells are all restricted by HLA-B alleles. Our findings suggest that the four epitopes in CFP10 recognized by CD8⁺ T cells might be of importance for the development of Mtb peptide-based vaccines and for improved diagnosis of TB in humans.     

ATM Influences the Efficiency of TCRβ Rearrangement,Subsequent TCRβ-Dependent T Cell Development,and Generation of the Pre-Selection TCRβ CDR3 Repertoire

Generation and resolution of DNA double-strand breaks is required to assemble antigen-specific receptors from the genes encoding V, D, and J gene segments during recombination. The present report investigates the requirement for ataxia telangiectasia-mutated (ATM) kinase, a component of DNA double-strand break repair, during TCRβ recombination and in subsequent TCRβ-dependent repertoire generation and thymocyte development. CD4⁻CD8⁻ double negative stage 2/3 thymocytes from ATM-deficient mice have both an increased frequency of cells with DNA break foci at TCRβ loci and reduced Vβ-DJβ rearrangement. Sequencing of TCRβ complementarity-determining region 3 demonstrates that ATM-deficient CD4⁺CD8⁺ double positive thymocytes and peripheral T cells have altered processing of coding ends for both in-frame and out-of-frame TCRβ rearrangements, providing the unique demonstration that ATM deficiency alters the expressed TCRβ repertoire by a selection-independent mechanism. ATMKO thymi exhibit a partial developmental block in DN cells as they negotiate the β-selection checkpoint to become double negative stage 4 and CD4⁺CD8⁺ thymocytes, resulting in reduced numbers of CD4⁺CD8⁺ cells. Importantly, expression of a rearranged TCRβ transgene substantially reverses this defect in CD4⁺CD8⁺ cells, directly linking a requirement for ATM during endogenous TCRβ rearrangement to subsequent TCRβ-dependent stages of development. These results demonstrate that ATM plays an important role in TCRβ rearrangement, generation of the TCRβ CDR3 repertoire, and efficient TCRβ-dependent T cell development.     

Synergistic effect of Abraxane that combines human IL15 fused with an albumin‐binding domain on murine models of pancreatic ductal adenocarcinoma

Nab‐paclitaxel (Abraxane), which is a nanoparticle form of albumin‐bound paclitaxel, is one of the standard chemotherapies for pancreatic ductal adenocarcinoma (PDAC). This study determined the effect of Abraxane in combination with a fusion protein, hIL15‐ABD, on subcutaneous Panc02 and orthotopic KPC C57BL/6 murine PDAC models. Abraxane combined with hIL15‐ABD best suppressed tumour growth and produced a 40%–60% reduction in the tumour size for Panc02 and KPC, compared to the vehicle group. In the combination group, the active form of interferon‐γ (IFN‐γ)‐secreting CD8⁺ T cells and CD11b⁺CD86⁺ M1 macrophages in tumour infiltrating lymphocytes (TILs) were increased. In the tumour drainage lymph nodes (TDLNs) of the combination group, there was a 18% reduction in CD8⁺IFN‐γ⁺ T cells and a 0.47% reduction in CD4⁺CD25⁺FOXP3⁺ regulatory T cells, as opposed to 5.0% and 5.1% reductions, respectively, for the control group. Superior suppression of CD11b⁺GR‐1⁺ myeloid‐derived suppressor cells (MDSCs) and the induction of M1 macrophages in the spleen and bone marrow of mice were found in the combination group. Abraxane and hIL15‐ABD effectively suppressed NF‐κB‐mediated immune suppressive markers, including indoleamine 2,3‐dioxygenase (IDO), Foxp3 and VEGF. In conclusion, Abraxane combined with hIL15‐ABD stimulates the anticancer activity of effector cells, inhibits immunosuppressive cells within the tumour microenvironment (TME) of PDAC, and produces a greater inhibitory effect than individual monotherapies.     

Human Cytomegalovirus (HCMV)-Specific CD4+ and CD8+ T Cells Are Both Required for Prevention of HCMV Disease in Seropositive Solid-Organ Transplant Recipients

In solid-organ transplant recipients (SOTR) the protective role of human cytomegalovirus (HCMV)-specific CD4⁺, CD8⁺ and γδ T-cells vs. HCMV reactivation requires better definition. The aim of this study was to investigate the relevant role of HCMV-specific CD4⁺, CD8⁺ and γδ T-cells in different clinical presentations during the post-transplant period. Thirty-nine SOTR underwent virologic and immunologic follow-up for about 1 year after transplantation. Viral load was determined by real-time PCR, while immunologic monitoring was performed by measuring HCMV-specific CD4⁺ and CD8⁺ T cells (following stimulation with autologous HCMV-infected dendritic cells) and γδ T-cells by flow cytometry. Seven patients had no infection and 14 had a controlled infection, while both groups maintained CD4⁺ T-cell numbers above the established cut-off (0.4 cell/µL blood). Of the remaining patients, 9 controlled the infection temporarily in the presence of HCMV-specific CD8⁺ only, until CD4⁺ T-cell appearance; while 9 had to be treated preemptively due to a viral load greater than the established cut-off (3×10⁵ DNA copies/mL blood) in the absence of specific CD4⁺ T-cells. Polyfunctional CD8⁺ T-cells as well as Vδ2⁻ γδ T-cells were not associated with control of infection. In conclusion, in the absence of HCMV-specific CD4⁺ T-cells, no long-term protection is conferred to SOTR by either HCMV-specific CD8⁺ T-cells alone or Vδ2⁻ γδ T-cell expansion.