Allergen‐specific naïve and memory CD4+ T cells exhibit functional and phenotypic differences between individuals with or without allergy

Although allergen‐specific CD4⁺ T cells are detectable in the peripheral blood of both individuals with or without allergy, their frequencies and phenotypes within the memory as well as naïve repertoires are incompletely known. Here, we analyzed the DRB1*0401‐restricted responses of peripheral blood‐derived memory (CD4⁺CD45RO⁺) and naïve (CD4⁺CD45RA⁺) T cells from subjects with or without allergy against the immunodominant epitope of the major cow dander allergen Bos d 2 by HLA class II tetramers in vitro. The frequency of Bos d 2_127–142‐specific memory T cells in the peripheral blood‐derived cultures appeared to be higher in subjects with allergy than those without, whereas naïve Bos d 2_127–142‐specific T cells were detectable in the cultures of both groups at nearly the same frequency. Surprisingly, the TCR avidity of Bos d 2_127–142‐specific T cells of naïve origin, as assessed by the intensity of HLA class II tetramer staining, was found to be higher in individuals with allergy. Upon restimulation, long‐term Bos d 2_127–142‐specific T‐cell lines generated from both memory and naïve T‐cell pools from individuals with allergy proliferated more strongly, produced more IL‐4 and IL‐10, and expressed higher levels of CD25 but lower levels of CXCR3 than the T‐cell lines from individuals without allergy, demonstrating differences also at the functional level. Collectively, our current results suggest that not only the memory but also the naïve allergen‐specific T‐cell repertoires differ between individuals with or without allergy.     

Cytomegalovirus‐seropositivity has a profound influence on the magnitude of major lymphoid subsets within healthy individuals

Cytomegalovirus (CMV) infects most individuals and elicits a strong CMV‐specific immune response. We have studied the influence of CMV‐seropositivity on the size of lymphoid subsets in healthy donors and demonstrate that the virus substantially modulates the peripheral lymphoid pool. CD8⁺ T cell numbers are increased in all CMV‐seropositive individuals because of a striking 60% increment in the CD8⁺ T cell memory pool. The CD45RA⁺ resting memory pool is doubled after CMV infection and increases further with age. The magnitude of the naïve CD8⁺ T cell pool is dramatically reduced in CMV‐seropositive individuals at all ages, and this accelerates the physiological decline by approximately 40 years. The number of CD4⁺ effector memory T cells is increased in CMV‐seropositive individuals and is differentially accommodated by a reduction in the number of naïve and central memory CD4⁺ T cells in young and elderly donors respectively. CMV‐seropositivity also increases the total number of B cells in older donors and suppresses the number of CD5⁺ B cells. These data reveal that CMV has a profound influence on the immune system of all healthy individuals and add to growing concern regarding the clinical and immunomodulatory significance of CMV infection in healthy donors.     

Mixed functional characteristics correlating with TCR‐ligand koff‐rate of MHC‐tetramer reactive T cells within the naive T‐cell repertoire

The low frequency of antigen‐specific naïve T cells has challenged numerous laboratories to develop various techniques to study the naïve T‐cell repertoire. Here, we combine the generation of naïve repertoire‐derived antigen‐specific T‐cell lines based on MHC‐tetramer staining and magnetic‐bead enrichment with in‐depth functional assessment of the isolated T cells. Cytomegalovirus (CMV) specific T‐cell lines were generated from seronegative individuals. Generated T‐cell lines consisted of a variety of immunodominant CMV‐epitope‐specific oligoclonal T‐cell populations restricted to various HLA‐molecules (HLA‐A1, A2, B7, B8, and B40), and the functional and structural avidity of the CMV‐specific T cells was studied. Although all CMV‐specific T cells were isolated based on their reactivity toward a specific peptide‐MHC complex, we observed a large variation in the functional avidity of the MHC‐tetramer positive T‐cell populations, which correlated with the structural avidity measured by the recently developed Streptamer k_off‐rate assay. Our data demonstrate that MHC‐tetramer staining is not always predictive for specific T‐cell reactivity, and challenge the sole use of MHC‐tetramers as an indication of the peripheral T‐cell repertoire, independent of the analysis of functional activity or structural avidity parameters.     

Characterization of the human CD4+ T‐cell repertoire specific for major histocompatibility class I‐restricted antigens

While CD4⁺ T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class‐I restricted CD4⁺ T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer‐based enrichment approach allowing detection and isolation of scarce Ag‐specific T cells, we performed a systematic comparative analysis of HLA‐A*0201‐restricted CD4⁺ and CD8⁺ T‐cell lines directed against several immunodominant viral or tumoral antigens. CD4⁺ T cells directed against every peptide‐MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA‐A2⁺ target cells carrying the relevant epitopes. HLA‐A2‐restricted CD4⁺ T cells were seldom expanded in immune HLA‐A2⁺ donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide‐MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class‐I restricted T helper cells and high affinity TCR that could be used for adoptive T‐cell transfer‐ or TCR gene transfer‐based immunotherapies.     

IL‐15 is critical for the maintenance and innate functions of self‐specific CD8+ T cells

IL‐15 is a pleiotropic cytokine involved in host defense as well as autoimmunity. IL‐15‐deficient mice show a decrease of memory phenotype (MP) CD8⁺ T cells, which develop naturally in naïve mice and whose origin is unclear. It has been shown that self‐specific CD8⁺ T cells developed in male H‐Y antigen‐specific TCR transgenic mice share many similarities with naturally occurring MP CD8⁺ T cells in normal mice. In this study, we found that H‐Y antigen‐specific CD8⁺ T cells in male but not female mice decreased when they were crossed with IL‐15‐deficient mice, mainly due to impaired peripheral maintenance. The self‐specific TCR transgenic CD8⁺ T cells developed in IL‐15‐deficient mice showed altered surface phenotypes and reduced effector functions ex vivo. Bystander activation of the self‐specific CD8⁺ T cells was induced in vivo during infection with Listeria monocytogenes, in which proliferation but not IFN‐γ production was IL‐15‐dependent. These results indicated important roles for IL‐15 in the maintenance and functions of self‐specific CD8⁺ T cells, which may be included in the naturally occurring MP CD8⁺ T‐cell population in naïve normal mice and participate in innate host defense responses.     

Frequent occurrence of high affinity T cells against MELOE‐1 makes this antigen an attractive target for melanoma immunotherapy

We recently showed that the infusion of tumor infiltrating lymphocytes specific for the MELOE‐1 antigen was associated with a prolonged relapse‐free survival for HLA‐A2⁺ melanoma patients who received tumor infiltrating lymphocytes therapy. Here, we characterized the MELOE‐1/A2‐specific T‐cell repertoire in healthy donors and melanoma patients to further support an immunotherapy targeting this epitope. Using tetramer enrichment followed by multicolor staining, we found that MELOE‐1‐specific T cells were present in the blood of healthy donors and patients at similar frequencies (around 1 in 1×10⁵ CD8⁺ cells). These cells mainly displayed a naïve phenotype in 4/6 healthy donors and 3/6 patients, whereas high proportions of memory cells were observed in the remaining individuals of both groups. There was a recurrent usage of the Vα12.1 chain for 17/18 MELOE‐1‐specific T‐cell clones derived from healthy donors or patients, associated with diverse Vβ chains and V(D)J junctional sequences. All clones derived from melanoma patients (9/9) were reactive against the MELOE‐1_36–44 peptide and against HLA‐A2⁺ melanoma cell lines. This study documents the existence of a large TCR repertoire specific for the MELOE‐1/A2 epitope and its capacity to give rise to antitumor CTL that supports the development of immunotherapies targeting this epitope.     

Memory B cells from a subset of treatment‐naïve relapsing‐remitting multiple sclerosis patients elicit CD4+ T‐cell proliferation and IFN‐γ production in response to myelin basic protein and myelin oligodendrocyte glycoprotein

Recent evidence suggests that B‐ and T‐cell interactions may be paramount in relapsing‐remitting MS (RRMS) disease pathogenesis. We hypothesized that memory B‐cell pools from RRMS patients may specifically harbor a subset of potent neuro‐APC that support neuro‐Ag reactive T‐cell proliferation and cytokine secretion. To test this hypothesis, we compared CD80 and HLA‐DR expression, IL‐10 and lymphotoxin‐α secretion, neuro‐Ag binding capacity, and neuro‐Ag presentation by memory B cells from RRMS patients to naïve B cells from RRMS patients and to memory and naïve B cells from healthy donors (HD). We identified memory B cells from some RRMS patients that elicited CD4⁺ T‐cell proliferation and IFN‐γ secretion in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Notwithstanding the fact that the phenotypic parameters that promote efficient Ag presentation were observed to be similar between RRMS and HD memory B cells, a corresponding capability to elicit CD4⁺ T‐cell proliferation in response to myelin basic protein and myelin oligodendrocyte glycoprotein was not observed in HD memory B cells. Our results demonstrate for the first time that the memory B‐cell pool in RRMS harbors neuro‐Ag specific B cells that can activate T cells.     

CD5 levels define functionally heterogeneous populations of naïve human CD4+ T cells

Studies in murine models show that subthreshold TCR interactions with self-peptide are required for thymic development and peripheral survival of naïve T cells. Recently, differences in the strength of tonic TCR interactions with self-peptide, as read-out by cell surface levels of CD5, were associated with distinct effector potentials among sorted populations of T cells in mice. However, whether CD5 can also be used to parse functional heterogeneity among human T cells is less clear. Our study demonstrates that CD5 levels correlate with TCR signal strength in human naïve CD4⁺ T cells. Further, we describe a relationship between CD5 levels on naïve human CD4⁺ T cells and binding affinity to foreign peptide, in addition to a predominance of CD5^hi T cells in the memory compartment. Differences in gene expression and biases in cytokine production potential between CD5^lo and CD5^hi naïve human CD4⁺ T cells are consistent with observations in mice. Together, these data validate the use of CD5 surface levels as a marker of heterogeneity among human naïve CD4⁺ T cells with important implications for the identification of functionally biased T- cell populations that can be exploited to improve the efficacy of adoptive cell therapies.     

High-frequency and adaptive-like dynamics of human CD1 self-reactive T cells

CD1 molecules present lipid antigens to T cells. An intriguing subset of human T cells recognize CD1‐expressing cells without deliberately added lipids. Frequency, subset distribution, clonal composition, naïve‐to‐memory dynamic transition of these CD1 self‐reactive T cells remain largely unknown. By screening libraries of T‐cell clones, generated from CD4⁺ or CD4^?CD8^? double negative (DN) T cells sorted from the same donors, and by limiting dilution analysis, we find that the frequency of CD1 self‐reactive T cells is unexpectedly high in both T‐cell subsets, in the range of 1/10–1/300 circulating T cells. These T cells predominantly recognize CD1a and CD1c and express diverse TCRs. Frequency comparisons of T‐cell clones from sorted naïve and memory compartments of umbilical cord and adult blood show that CD1 self‐reactive T cells are naïve at birth and undergo an age‐dependent increase in the memory compartment, suggesting a naïve/memory adaptive‐like population dynamics. CD1 self‐reactive clones exhibit mostly Th1 and Th0 functional activities, depending on the subset and on the CD1 isotype restriction. These findings unveil the unanticipated relevance of self‐lipid T‐cell response in humans and clarify the basic parameters of the lipid‐specific T‐cell physiology.     

Ex vivo monitoring of human cytomegalovirus‐specific CD8+ T‐Cell responses using the QuantiFERON®‐CMV assay in allogeneic hematopoietic stem cell transplant recipients attending an Irish hospital

Reconstitution of human cytomegalovirus (HCMV) T‐cell immunity is crucial in hematopoietic stem cell transplant (HSCT) recipients. The QuantiFERON®‐CMV assay for cellular HCMV‐specific immunity was evaluated in allogeneic HSCT recipients (n = 43) and patients with hematological malignancies (n = 29) attending a tertiary‐care Irish hospital. An intracellular cytokine (ICC) assay correlated with the QuantiFERON®‐CMV assay. Although there was agreement between HCMV seropositivity and QuantiFERON®‐CMV assay, six HCMV seropositive immunosuppressed patients with hematological malignancy had negative QuantiFERON®‐CMV results. The 43 HSCT recipients were classified as high risk (D^?/R⁺) (n = 18), intermediate risk (D⁺/R⁺ and D⁺/R^?) (n = 17), and low risk (D^?/R^?) (n = 8). During episodes of HCMV DNAemia no evidence of HCMV‐specific immunity was found using the QuantiFERON®‐CMV assay. Furthermore, the recovery of HCMV‐specific CD8⁺ T‐cell responses in high‐risk seropositive recipients of matched unrelated donors was severely delayed, a mean of 200 (SD = 117) days compared to 58 (SD = 23) days for sibling donors (P ≤ 0.028). In addition, three patients with late HCMV infection (infection >100 days post‐transplant) had delayed reconstitution of HCMV‐specific CD8⁺ T cells. Interestingly, two recipients (R⁺/D^?) developed rapid immune reconstitution by days 15 and 36 post‐HSCT, suggesting HCMV‐specific T‐cell lymphopoiesis of recipient origin. Levels of CD8⁺ T‐cell immunity in HCMV seropositive HSCT recipients were lowest following HSCT. A high number (33%) of indeterminate results was observed immediately after transplantation. Patients with indeterminate QuantiFERON®‐CMV results had low levels of HCMV‐specific CD8⁺ T cells. J. Med. Virol. 82:433–440, 2010. © 2010 Wiley‐Liss, Inc.     

Tolerogenic dendritic cells impede priming of naïve CD8+ T cells and deplete memory CD8+ T cells

Type 1 diabetes is a T‐cell‐mediated autoimmune disease in which autoreactive CD8⁺ T cells destroy the insulin‐producing pancreatic beta cells. Vitamin D3 and dexamethasone‐modulated dendritic cells (Combi‐DCs) loaded with islet antigens inducing islet‐specific regulatory CD4⁺ T cells may offer a tissue‐specific intervention therapy. The effect of Combi‐DCs on CD8⁺ T cells, however, remains unknown. To investigate the interaction of CD8⁺ T cells with Combi‐DCs presenting epitopes on HLA class I, naive, and memory CD8⁺ T cells were co‐cultured with DCs and proliferation and function of peptide‐specific T cells were analyzed. Antigen‐loaded Combi‐DCs were unable to prime naïve CD8⁺ T cells to proliferate, although a proportion of T cells converted to a memory phenotype. Moreover, expansion of CD8⁺ T cells that had been primed by mature monocyte‐derived DCs (moDCs) was curtailed by Combi‐DCs in co‐cultures. Combi‐DCs expanded memory T cells once, but CD8⁺ T‐cell numbers collapsed by subsequent re‐stimulation with Combi‐DCs. Our data point that (re)activation of CD8⁺ T cells by antigen‐pulsed Combi‐DCs does not promote, but rather deteriorates, CD8⁺ T‐cell immunity. Yet, Combi‐DCs pulsed with CD8⁺ T‐cell epitopes also act as targets of cytotoxicity, which is undesirable for survival of Combi‐DCs infused into patients in therapeutic immune intervention strategies.     

T cell receptor alpha variable 12‐2 bias in the immunodominant response to Yellow fever virus

The repertoire of human αβ T‐cell receptors (TCRs) is generated via somatic recombination of germline gene segments. Despite this enormous variation, certain epitopes can be immunodominant, associated with high frequencies of antigen‐specific T cells and/or exhibit bias toward a TCR gene segment. Here, we studied the TCR repertoire of the HLA‐A*0201‐restricted epitope LLWNGPMAV (hereafter, A2/LLW) from Yellow Fever virus, which generates an immunodominant CD8⁺ T cell response to the highly effective YF‐17D vaccine. We discover that these A2/LLW‐specific CD8⁺ T cells are highly biased for the TCR α chain TRAV12‐2. This bias is already present in A2/LLW‐specific naïve T cells before vaccination with YF‐17D. Using CD8⁺ T cell clones, we show that TRAV12‐2 does not confer a functional advantage on a per cell basis. Molecular modeling indicated that the germline‐encoded complementarity determining region (CDR) 1α loop of TRAV12‐2 critically contributes to A2/LLW binding, in contrast to the conventional dominant dependence on somatically rearranged CDR3 loops. This germline component of antigen recognition may explain the unusually high precursor frequency, prevalence and immunodominance of T‐cell responses specific for the A2/LLW epitope.     

Cytomegalovirus drives Vδ2neg γδ T cell inflation in many healthy virus carriers with increasing age

Cytomegalovirus (CMV) usually causes lifelong asymptomatic infection, but over time can distort immune profiles. Recent reports describe selective expansion of Vδ2^neg γδ T cells in healthy and immunocompromised CMV carriers. Having shown previously that virus‐specific CD8⁺ and CD4⁺ T cell responses are increased significantly in elderly CMV carriers, probably driven by chronic stimulation, we hypothesized that Vδ2^neg γδ T cells may also be expanded with age. Our results show that Vδ2^neg γδ T cells are increased significantly in CMV‐seropositive healthy individuals compared to CMV‐seronegative controls in all age groups. The differences were most significant in older age groups (P < 0·0001). Furthermore, while Vδ2^neg γδ T‐ cells comprise both naive and memory cells in CMV‐seronegative donors, highly differentiated effector memory cells are the dominant phenotype in CMV carriers, with naive cells reduced significantly in numbers in CMV‐seropositive elderly. Although phenotypically resembling conventional CMV‐specific T cells, Vδ2^neg γδ T cells do not correlate with changes in magnitude of CMV‐specific CD4⁺ or CD8⁺ T cell frequencies within those individuals, and do not possess ex‐vivo immediate effector function as shown by CMV‐specific CD4⁺ and CD8⁺ T cells. However, after short‐term culture, Vδ2^neg γδ T cells demonstrate effector T cell functions, suggesting additional requirements for activation. In summary, Vδ2^neg γδ T cells are expanded in many older CMV carriers, demonstrating a further level of lymphocyte subset skewing by CMV in healthy individuals. As others have reported shared reactivity of Vδ2^neg γδ T cells towards tumour cells, the composition of γδ T cell subsets may also have implications for risk of developing cancer in elderly people.     

Positive selection of self‐antigen‐specific CD8+ T cells by hematopoietic cells

In contrast to thymic epithelial cells, which induce the positive selection of conventional CD8⁺ T cells, hematopoietic cells (HCs) select innate CD8⁺ T cells whose Ag specificity is not fully understood. Here we show that CD8⁺ T cells expressing an H‐Y Ag‐specific Tg TCR were able to develop in mice in which only HCs expressed MHC class I, when HCs also expressed the H‐Y Ag. These HC‐selected self‐specific CD8⁺ T cells resemble innate CD8⁺ T cells in WT mice in terms of the expression of memory markers and effector functions, but are phenotypically distinct from the thymus‐independent CD8⁺ T‐cell population. The peripheral maintenance of H‐Y‐specific CD8⁺ T cells required presentation of the self‐Ag and IL‐15 on HCs. HC‐selected CD8⁺ T cells in mice lacking the Tg TCR also showed these features. Furthermore, by using MHC class I tetramers with a male Ag peptide, we found that self‐Ag‐specific CD8⁺ T cells in TCR non‐Tg mice could develop via HC‐induced positive selection, supporting results obtained from H‐Y TCR Tg mice. These findings indicate the presence of self‐specific CD8⁺ T cells that are positively selected by HCs in the peripheral T‐cell repertoire.     

Freeze-thaw lysates of Plasmodium falciparum-infected red blood cells induce differentiation of functionally competent regulatory T cells from memory T cells

In addition to naturally occurring regulatory T (nTreg) cells derived from the thymus, functionally competent Treg cells can be induced in vitro from peripheral blood lymphocytes in response to TCR stimulation with cytokine costimulation. Using these artificial stimulation conditions, both naïve as well as memory CD4⁺ T cells can be converted into induced Treg (iTreg) cells, but the cellular origin of such iTreg cells in vivo or in response to more physiologic stimulation with pathogen‐derived antigens is less clear. Here, we demonstrate that a freeze/thaw lysate of Plasmodium falciparum schizont extract (PfSE) can induce functionally competent Treg cells from peripheral lymphocytes in a time‐ and dose‐dependent manner without the addition of exogenous costimulatory factors. The PfSE‐mediated induction of Treg cells required the presence of nTreg cells in the starting culture. Further experiments mixing either memory or naïve T cells with antigen presenting cells and CFSE‐labeled Treg cells identified CD4⁺CD45RO⁺CD25^? memory T cells rather than Treg cells as the primary source of PfSE‐induced Treg cells. Taken together, these data suggest that in the presence of nTreg cells, PfSE induces memory T cells to convert into iTreg cells that subsequently expand alongside PfSE‐induced effector T cells.     

Paths to expansion: Differential requirements of IRF4 in CD8+ T‐cell expansion driven by antigen and homeostatic cytokines

Interferon regulatory factor 4 (IRF4) regulates the clonal expansion and metabolic activity of activated T cells, but the precise context and mechanisms of its function in these processes are unclear. In this issue of the European Journal of Immunology, Miyakoda et al. [Eur. J. Immunol. 2018. 48 : 1319–1328] show that IRF4 is required for activation and expansion of naïve and memory CD8⁺ T cells driven by T‐cell receptor (TCR) signaling, but dispensable for memory CD8⁺ T‐cell maintenance and homeostatic proliferation driven by homeostatic cytokines. The authors show that the function of IRF4 in CD8⁺ T‐cell expansion is partially dependent upon activation of the PI3K/AKT pathway through direct or indirect attenuation of PTEN expression. These data shed light upon the differential intracellular pathways required for naïve and memory T cells to respond to self‐antigens and/or homeostatic cytokines, and highlight the potential translational relevance of these findings in the context of immune reconstitution such as following allogeneic stem cell transplantation.     

Establishment and Operation of a Third-Party Virus-Specific T Cell Bank within an Allogeneic Stem Cell Transplant Program

Hematopoietic stem cell transplantation (HSCT) donor–generated virus-specific T cells (VSTs) can provide effective treatment for viral infection post-HSCT but are not readily accessible to all patients. Off-the-shelf cryopreserved VSTs suitable for treatment of multiple patients are an attractive alternative. We generated a bank of 17 cytomegalovirus (CMV)–, 14 Epstein-Barr virus (EBV)–, and 15 adenovirus (AdV)–specific T cell products from 30 third-party donors. Donors were selected for expression of 6 core HLA antigens expressed at high frequency in the local transplant population. T cells were generated by co-culturing venous blood or mobilized hematopoietic stem cell (HSC)–derived mononuclear cells with monocyte-derived dendritic cells pulsed with overlapping peptides covering CMV pp65, AdV5 hexon, or EBV BZLF1/LMP2A/EBNA1 proteins. Addition of a CD14⁺ selection step instead of plate adherence to isolate monocytes before culture initiation significantly improved expansion in cultures from HSC material. Phenotyping showed the CD8⁺ subset to have significantly higher numbers of terminal effector T cells (CD45RA⁺62L^?) and lower numbers of effector memory T cells (CD45RA^?62L^?) when compared with the CD4⁺ subset. Increased expression of the immunoinhibitory markers PD-1 and TIM-3 was noted on CD4⁺ but not CD8⁺ cells when compared with the control group. VST showed antiviral activity restricted through a variety of common HLAs, and modelling suggested a suitably HLA-matched product would be available for >90% of HSCT patients. Only a small number of carefully selected third-party donors are required to generate a VST bank of broad coverage, indicating the feasibility of local banking integrated into existing allogeneic HSCT programs.