Tfh CD4 Research Articles

A Distinct T Follicular Helper Cell Subset Infiltrates the Brain in Murine Neuropsychiatric Lupus.

Neuropsychiatric symptoms in systemic lupus erythematosus (SLE) are not uncommon, yet the mechanisms underlying disease initiation and progression in the brain are incompletely understood. Although the role of T cells in other lupus target organs such as the kidney is well defined, which T cells contribute to the pathogenesis of neuropsychiatric SLE is not known. The present study was aimed at characterizing the CD4 T cell populations that are present in the choroid plexus (CP) of MRL/MpJ-faslpr mice, the primary site of brain infiltration in this classic lupus mouse model which exhibits a prominent neurobehavioral phenotype. T cells infiltrating the CP of MRL/MpJ-faslpr mice were characterized and subset identification was done by multiparameter flow cytometry. We found that the infiltrating CD4 T cells are activated and have an effector phenotype. Importantly, CD4 T cells have a T follicular helper cell (TFH) like phenotype, as evidenced by their surface markers and signature cytokine, IL-21. In addition, CD4 TFH cells also secrete significant levels of IFN-γ and express Bcl-6, thereby conforming to a potentially pathogenic T helper population that can drive the disease progression. Interestingly, the regulatory axis comprising CD4 T regulatory cells is diminished. These results suggest that accumulation of CD4 TFH in the brain of MRL/MpJ-faslpr mice may contribute to the neuropsychiatric manifestations of SLE, and point to this T cell subset as a possible novel therapeutic candidate.

Host Expression of the CD8 Treg/NK Cell Restriction Element Qa-1 is Dispensable for Transplant Tolerance

Disruption of the non-classical Major Histocompatibility Complex (MHC) Ib molecule Qa-1 impairs CD8 Treg and natural killer (NK) cell function and promotes a lupus-like autoimmune disease. This immune perturbation would be expected to enhance anti-transplant responses and impair tolerance induction, but the effect of Qa-1 deficiency on the transplant response has not been previously reported. Qa-1 deficiency enhanced CD4 TFH and germinal center (GC) B cell numbers in naïve mice and hastened islet allograft rejection. Despite enhanced immunity in B6.Qa-1−/− mice, these mice did not generate an excessive primary CD4 TFH cell response nor an enhanced alloantibody reaction. Both CD8 Tregs and NK cells, which often regulate other cells through host Qa-1 expression, were targets of anti-CD45RB therapy that had not been previously recognized. However, B6.Qa-1−/− mice remained susceptible to anti-CD45RB mediated suppression of the alloantibody response and transplant tolerance induction to mismatched islet allografts. Overall, despite enhanced immunity as demonstrated by augmented CD4 TFH/GC B cell numbers and hastened islet allograft rejection in naïve 12-week old Qa-1 deficient mice, the CD8 Treg/NK cell restriction element Qa-1 does not regulate the primary cellular or humoral alloresponse and is not required for long-term transplant tolerance.

Follicular CD8 T cells in autoantibody-mediated disease.

Abstract The role of CD8 T cells, unlike CD4 T cells, in antibody-mediated autoimmune disease remains largely unexplored. CD8 T cells act both in a cytotoxic CD8 T cell role and helper-like role during autoimmunity. CD4 T follicular cells (Tfh), interact with B cells to promote proliferation, activation and antibody class switching. In autoimmunity, dysregulated self-reactive CD4 Tfh can promote autoantibody production. We have identified a novel population of CD8 T cells within the germinal center that resembles CD4 Tfh cells during systemic autoantibody-mediated disease. IL-2 deficient (IL-2-KO) mice develop systemic autoantibody-mediated disease including autoantibodies against red blood cells (RBC) resulting in autoimmune hemolytic anemia. We show that the absence of either CD4 or CD8 T cells prevents the development of anti-RBC antibodies and reduces lymphoproliferation in IL-2-KO mice. RNA-sequencing of IL-2-KO CD8 T cells revealed an increase in the expression of genes associated with CD4 Tfh lineages (Bcl-6, CXCR5, PD-1, ICOS and IL-21) compared with wild type (WT) CD8 T cells. Flow cytometry of expanded CXCR5+PD-1+ CD4 Tfh cells and CXCR5+PD-1+ CD8 T follicular (Tfc) cells confirms the protein expression of these Tfh markers and the production of IL-21 in IL-2-KO late stage disease (day 18–21). In vitro culture of IL-2-KO CD8 Tfc cells induced antibody class switching in WT B cells at levels similar to that induced by adjuvant-induced WT CD4 Tfh cells. In comparison to CXCR5- PD-1-CD8 T cells, CD8 Tfc cells expressed higher levels of CD40L. Our results indicate that CD8 T cells contribute to autoimmune disease in part through differentiation into follicular T cells that function to induce B cell differentiation and antibody class-switch.

Germinal Center Immune Dynamics in Secondary Lymphoid Organs after Influenza Vaccination in Children

Abstract Introduction The germinal center immune dynamics leading to protective antibody responses after vaccination in humans remains largely unknown. Investigation of such dynamics would provide valuable information regarding the cellular and molecular mechanisms governing the generation of these responses and lead to novel vaccine strategies Materials and methods Paraffin embedded tonsil tissue sections were analyzed by multiplexed confocal microscopy assays and Histo-cytometry. The phenotype of tonsil derived cells was analyzed with polychromatic flow cytometry, flu-specific B cell-responses were detected by ELISPOT to H1N1, H3N2, and B strain and plasmas analyzed with a Luminex multiplex assay. Results 36 children (24 vaccinated with 2015/2016 seasonal influenza vaccine and 12 controls) scheduled for elective tonsillectomy were included. Flow cytometry and Histo-Cytometry analysis showed that vaccination induced the frequency of tonsil CD4 Tfh, especially the PD-1hiCD57lo ones. This profile was associated with a significant reduction of potential tissue suppressor CD4 T cells and positively correlated with all flu-antigen specific B cells. An induction of IL-4R on plasma cells was found after vaccination implying a further mobilization of these cells. Among the circulating cytokines/chemokines tested, CXCL-13 was selectively induced after vaccination, in line with the increased frequency of CD4 Tfh found. Conclusions Considered that children are continuously scheduled for tonsillectomy, tonsils could represent a valuable and approachable secondary lymphoid organ for the investigation of germinal center reactions after vaccination compared to lymph node not easily accessible in this population.

Competition within the virus-specific CD4 T-cell pool limits the T follicular helper response after influenza infection.

CD4 T follicular helper cells (TFH) are critical in the generation of potent and long-lived B-cell responses after viral infection. However, the factors that dictate the generation and maintenance of these cells are not fully understood. Here we use adoptive transfer of OTII T-cell receptor transgenic CD4 T cells, followed by infection with recombinant influenza A virus (IAV), as a means of identifying and tracking virus-specific CD4(+) T-cell responses. We show that T-cell competition within the virus-specific CD4 T-cell pool induced by IAV infection limits the proliferation and differentiation of IAV-specific CD4(+) TFH responses. In particular, increased T-cell competition for antigen results in a diminished IAV-specific TFH CD4 T-cell responses, particularly germinal center TFH responses. Strikingly, competition in the form of preexisting cellular immunity generated by heterosubtypic IAV immunization limits de novo CD4 T-cell responses in secondary lymphoid tissue. Taken together, these data show a profound linkage between antigen availability and promotion of TFH CD4(+) T-cell responses in response to infection. These data suggest that competition within the CD4 T-cell pool limits TFH responses and may be an important regulatory mechanism for controlling immunity.

Dysregulated CD8 T cells resemble Tfh-like cells and promote B cell activation during autoimmunity.

Abstract T follicular helper (Tfh) cells provide essential help for the development of antigen specific antibodies during normal and autoreactive immune responses. IL-2-deficient (KO) mice suffer from excessive lymphoproliferation and spontaneous, systemic autoimmune disease. Depletion of CD4 T cells in IL-2-KO mice eliminates the generation of RBC-specific antibodies, augmenting survival. Surprisingly, depletion of CD8 T cells in IL-2-KO mice also reduces disease and prolongs survival. Several groups have demonstrated a protective role for CD8 T cells in inflammation and autoimmunity, while others have suggested a pathogenic role. However the mechanisms of CD8 T cell influence on autoimmune disease are largely unknown. We have identified a new CD8 Tfh-like population in an autoimmune setting. RNA-sequencing of day 12 IL-2-KO CD8 T cells revealed an increase in the expression of genes associated with the CD4 Tfh lineage (Bcl-6, CXCR5, PD-1, ICOS and IL-21) compared with wild-type CD8 T cells. Flow cytometry confirmed the presence of expanded CD4 Tfh and CD8 Tfh-like cells, and the production of IL-21 in IL-2-KO mice. Germinal center tissue staining revealed localization of both CD4 and CD8 T cells to the germinal center during autoimmunity. In vitro, IL-2-KO CD8 T cells induced proliferation and activation of B cells. Our results indicate that autoreactive CD8 T cells contribute to the autoimmune disease in part through dysregulated follicular-like differentiation and function. NIH funding R00HL090706.

Sepsis compromises primary B cell-mediated responses

Abstract Sepsis is the leading cause of death in non-coronary ICU’s, and one hallmark of sepsis patients is sustained immune suppression. Septic patients exhibit compromised innate and adaptive immunity, making them highly susceptible to secondary infections. Using the cecal ligation and puncture (CLP) model of sepsis, we recently showed the recovery of CD4 T cells from CLP-induced lymphopenia is accompanied by alterations in the composition and function of the Ag-specific CD4 T cell repertoire. These data led us to hypothesize that CD4 T cell-dependent B cell responses would also be impaired after CLP. Using the same CLP model, we immunized sham (control) and CLP mice with PE-conjugated 2W1S56 peptide 30 d after surgery, and then we quantitated the number of 2W1S-specific CD4 T cells and PE-specific B cells 7 d later. Compared to sham mice, CLP-treated mice immunized with PE-2W1S56 had a sustained reduction in number of 2W1S-specific Tfh CD4 T cells and naïve/memory PE-specific B cells after CLP. Next, we immunized sham and CLP mice with TNP-KLH plus either CpG or alum early (2 d) or late (30 d) after surgery to induce Th1 or Th2 Ab responses, respectively. We observed compromised production of anti-TNP Ab in mice immunized with either TNP-KLH/CpG or alum early (2 d) after CLP. Importantly, Ab production remained reduced in mice immunized with TNP-KLH/alum late (30 d) after CLP. Together, our data show CLP-induced sepsis impacts humoral immunity by affecting the number and function of Ag-specific B cells and CD4 Tfh cells. Moreover, these data suggest potential for defects in humoral immunity to newly introduced Ag (e.g., vaccines) in sepsis survivors long after the septic event has resolved.

Intricate interplay of cytokines determine the progression of SLE-like disease in BXSB.Yaa mice (BA12P.104)

Abstract Systemic lupus erythematosus (SLE) is a complex multi-organ disease that affects mostly females. It is caused by several environmental, genetic and stochastic factors. BXSB.Yaa is a recombinant inbred strain of mice with major manifestations that parallel those of human SLE, thereby making it a model system to investigate genetic and non-genetic determinants of disease initiation and progression. The Yaa mutation in male results due to translocation of a 4Mb chromosomal region, including Tlr7 gene, from X to the Y chromosome, making them susceptible to disease. The disease pathogenesis involves an intricate interplay of key cytokines such as IL-21, IL-6, Type I interferon (IFN) and IL-10. Enhanced expression of IL-21 by CD4-T (Tfh) cells expands autoreactive B cells that differentiate into plasmablasts and secrete autoantibodies. IL21 also promotes the development of CD8-T cells and NK cells which retard early disease development. Gene expression profiles of cells from young IL-21R deficient BXSB.Yaa mice revealed an “IFN signature” similar to human SLE females. Purified plasmacytoid dendritic cells (pDCs) from male mice produced higher levels of Type-I IFN than females. Type I IFN further stimulates IL6 producing B cells that drives differentiation of CD4 Tfh cells. IL6 also contributes to accelerated mortality in BXSB.Yaa mice. Moreover, BXSB.Yaa mice express IL10 and ablation of IL10 in these mice results in accelerated mortality.

CD8+ /ICOS+ /CXCR5+ Tumor Infiltrating T-CELLS (CD8fc) Sharing Functional Similarities With TFH CELLS ARE Present In Classical Hodgkin'S Lymphoma Tissues Displaying A Specific “MIXED NODULARITY” Pattern

We have previously reported that some classical Hodgkin's Lymphoma (cHL) tissues display a gene signature evocative of a Th1 immune reaction. In order to better characterize this process, immune cell subsets were isolated from cHL tissue samples (n=21) using a powerful multicolor flow cytometry method, in parallel with cell sorting. Fresh tissue samples from follicular B cell lymphoma (FL, n=8), diffuse large cell B cell lymphoma (n=8) and reactive lymphadenitis (n= 5) were used as controls.In 4 cLH cases, we observed a significant proportion of activated CD8+ T-cells expressing ICOS and CXCR5 at high levels. The presence of either CD8+/ICOS+/CXCR5- T cells or CD8+/ICOS +/ CXCR5+ T-cells was a specific feature of HL tissues since it was absent from B-cell lymphomas, T-cell lymphomas and reactive tissues. In contrast, CD8+/CXCR5+ T-cells were found not only in cHL, but also in most other samples analyzed.Further phenotypic characterization showed that the CD8+/ICOS +/ CXCR5+ T cells expressed markers associated with CD4 TFH cells, like PD1, BTLA, bcl-6 and IL-21. Under stimulation, they expressed only low levels of IFNG, granzyme B and perforin, and thus do not fulfill the criteria of activated cytotoxic effectors. Co-culture experiments showed a dramatic enhancement of CD86 expression on stimulated B-cells in contact with CD8+/ICOS +/ CXCR5+ T cells. This effect was similarly observed after co-culture with CD4+TFH cells. The 4 cHL cases associated with CD8+/ICOS +/ CXCR5+ T-cells contained CD30+ CD15+ EBV+ Reed Sternberg cells (RSC). They were characterized a nodular non-sclerotic pattern reminiscent of the nodular lymphocyte-rich classical HL (NLRCHL) subtype, but also displayed a specific “mixed nodularity” feature. Various nodules were indeed observed, including reactive germinal centers (GC) partly colonized by RSC co-localizing with CD8+/ICOS+ T-cells, suggesting an early GC invasion triggering an intra-follicular CD8 T-cell reaction. Other nodules were composed of a high number of RS cells admixed with numerous CD8+/ICOS+ T-cells. This “mixed nodularity” pattern was absent in the other HL cases.Altogether, our results point out a previously unrecognized intra- follicular CD8 T-cell subset sharing phenotypic and functional features with CD4 TFH, that we have thus considered as putative “follicular cytotoxic” CD8 T-cells (TFC). This cell subset appears to be specifically associated with EBV+ cHL tissues with unusual histo-phenotypic features, which may probably reflect a strong CD8 activation process. Disclosures:No relevant conflicts of interest to declare.

CD8+ Treg - From Mouse To Man

T cell-mediated regulation of the immune response to self and foreign antigens is essential to maintain immune homeostasis and prevent autoimmune tissue destruction. The majority of studies addressing this issue, both in mice and humans, have focused on the contribution of CD4+CD25+FOXP3+ regulatory T cells. The contribution of regulatory CD8+ T cells has not been appreciated until recently. Recent studies have identified a small subset of IL-15 dependent CD8+ regulatory T cells (Treg) that is essential for maintenance of self- tolerance and prevention of autoimmune disease in mice. Expression of a triad of cell surface markers – CD44+CD122+Ly49+ – has been used to distinguish and purify CD8+Treg (Kim et al., Nature 2010; Kim et al., PNAS 2011).Here we have defined the human homologue of CD8+ Treg. The Ly49 receptor, identified as a stable surface marker on CD8+ Treg, is a member of a multigenic/multiallelic receptor family recognizing classical MHC class I molecules. The functional homologue of murine Ly49 is the killer cell immunoglobulin-like receptor (KIR). Our analysis has revealed that, while expression of KIR subtype combinations appears to be stochastic and co-expression of these KIR receptors is random, the inhibitory KIR2DL2/3 and KIR3DL1 subtypes are dominantly expressed by human CD3+CD8+ T cells. Similar to murine CD8+ Treg, CD8+ T cells that express KIR2DL2/3 or KIR3DL1 also express CD44 and CD122. Moreover, consistent with murine CD8+ Treg, incubation with IL-15 results in activation and proliferation of KIR+CD8+ T cells that maintain a stable surface phenotype. Gene array analyses in mice has indicated that Helios, a highly conserved zinc finger transcription factor and member of the Ikaros family of transcription factors, is expressed by CD44+CD122+Ly49+ CD8+ Treg. Helios is involved in T cell development and expressed by ∼70% of FoxP3+CD4+ Treg but not by mature B cells, dendritic cells or myeloid cells. Our analyses identified a Helios+ subset in the CD8+ Treg population, which (compared to the Helios- subset) embodies many of the functional characteristics of CD8+ Treg in mice. Co-expression of Helios is also apparent in some KIR+CD8+ T cells in human samples. In mice, we have shown that CD8+ Treg target CD4 TFH cells and thus maintain self-tolerance. In vitro suppression assays revealed that KIR+CD8+ cells but not KIR–CD8+ cells confer inhibitory activity on CD4+CXCR5+ TFHtarget cells in humans.Taken together, our findings implicate KIR+CD8+ cells as the human homologue of murine CD8+ Treg, including expression of transcription factor Helios, responsiveness to IL-15 and suppression of CD4+ TFH cells. Understanding the genetic and biological features of this CD8+ T cell subset in humans opens the possibility of exploiting their regulatory activity for the development of immunotherapy in the context of autoimmune disease and cancer. Disclosures:No relevant conflicts of interest to declare.

HIV vaccine research and discovery in the nonhuman primates model

Here we highlight the latest advances in HIV vaccine concepts that will expand our knowledge on how to elicit effective acquisition-prevention and/or control of simian immunodeficiency virus (SIV) replication in the nonhuman primate (NHP) model. In the context of the promising analyses from the RV144 Thai Trial and the effective control of SIV replication exerted by rhCMV-(SIV) elicited EM CD8 T cells, the HIV field has recently shifted toward vaccine concepts that combine protection from acquisition with effective control of SIV replication. Current studies in the NHP model have demonstrated the efficacy of HIV-neutralizing antibodies via passive transfer, the potential importance of the CD4 Tfh subset, the ability to effectively model the RV144 vaccine trial and the capacity of an Ad26 prime and modified vaccinia Ankara virus boost to elicit Env-specific antibody and cellular responses that both limit acquisition and control heterologous SIVmac251 challenge. The latest work in the NHP model suggests that the next generation HIV-1 vaccines should aim to provoke a comprehensive adaptive immune response for both prevention of SIV acquisition as well as control of replication in breakthrough infection.

Donor CD8 T cell activation is critical for greater renal disease severity in female chronic graft-vs.-host mice and is associated with increased splenic ICOShi host CD4 T cells and IL-21 expression

Lupus-like renal disease in DBA/2-into-F1 (DBA --> F1) mice is driven by donor CD4 T cells and is more severe in females. Donor CD8 T cells have no known role. As expected, we observed that females receiving unfractionated DBA splenocytes (CD8 intact --> F1) exhibited greater clinical and histological severities of renal disease at 13 weeks compared to males. Surprisingly, sex-based differences in renal disease severity were lost in CD8 depleted --> F1 mice due to an improvement in females and a worsening in males. CD8 intact --> F1 female mice exhibited significantly greater donor and host effector (CD44(hi), CD62L(lo)) CD4 T cells and ICOS(hi) CD4 T follicular helper cells than males. CD8 depleted --> F1 female mice exhibited a reduction in the absolute numbers of host, but not donor CD4 Tfh cells and lost the significant increase in host CD4 effector cells vs. males. Greater female IL-21 expression, a product of Tfh cells, was seen in CD8 intact --> F1 and although reduced was still greater than male CD8 depleted --> F1 mice. Thus, donor CD8 T cells have a critical role in mediating sex-based differences in lupus renal disease severity possibly through greater host ICOS(hi) CD4 T cell involvement.