Single-positive CD4 Research Articles

Key Factors for Thymic Function and Development.

The thymus is the organ responsible for T cell development and the formation of the adaptive immunity function. Its multicellular environment consists mainly of the different stromal cells and maturing T lymphocytes. Thymus-specific progenitors of epithelial, mesenchymal, and lymphoid cells with stem cell properties represent only minor populations. The thymic stromal structure predominantly determines the function of the thymus. The stromal components, mostly epithelial and mesenchymal cells, form this specialized area. They support the consistent developmental program of functionally distinct conventional T cell subpopulations. These include the MHC restricted single positive CD4+ CD8- and CD4- CD8+ cells, regulatory T lymphocytes (Foxp3+), innate natural killer T cells (iNKT), and γδT cells. Several physiological causes comprising stress and aging and medical treatments such as thymectomy and chemo/radiotherapy can harm the thymus function. The present review summarizes our knowledge of the development and function of the thymus with a focus on thymic epithelial cells as well as other stromal components and the signaling and transcriptional pathways underlying the thymic cell interaction. These critical thymus components are significant for T cell differentiation and restoring the thymic function after damage to reach the therapeutic benefits.

ICAP-1 loss impairs CD8+ thymocyte development and leads to reduced marginal zone B cells in mice.

ICAP‐1 regulates β1‐integrin activation and cell adhesion. Here, we used ICAP‐1‐null mice to study ICAP‐1 potential involvement during immune cell development and function. Integrin α4β1‐dependent adhesion was comparable between ICAP‐1‐null and control thymocytes, but lack of ICAP‐1 caused a defective single‐positive (SP) CD8+ cell generation, thus, unveiling an ICAP‐1 involvement in SP thymocyte development. ICAP‐1 bears a nuclear localization signal and we found it displayed a strong nuclear distribution in thymocytes. Interestingly, there was a direct correlation between the lack of ICAP‐1 and reduced levels in SP CD8+ thymocytes of Runx3, a transcription factor required for CD8+ thymocyte generation. In the spleen, ICAP‐1 was found evenly distributed between cytoplasm and nuclear fractions, and ICAP‐1–/– spleen T and B cells displayed upregulation of α4β1‐mediated adhesion, indicating that ICAP‐1 negatively controls their attachment. Furthermore, CD3+‐ and CD19+‐selected spleen cells from ICAP‐1‐null mice showed reduced proliferation in response to T‐ and B‐cell stimuli, respectively. Finally, loss of ICAP‐1 caused a remarkable decrease in marginal zone B‐ cell frequencies and a moderate increase in follicular B cells. Together, these data unravel an ICAP‐1 involvement in the generation of SP CD8+ thymocytes and in the control of marginal zone B‐cell numbers.

A Murine Model of X-Linked Moesin-Associated Immunodeficiency (X-MAID) Reveals Defects in T Cell Homeostasis and Migration.

X-linked moesin associated immunodeficiency (X-MAID) is a primary immunodeficiency disease in which patients suffer from profound lymphopenia leading to recurrent infections. The disease is caused by a single point mutation leading to a R171W amino acid change in the protein moesin (moesinR171W). Moesin is a member of the ERM family of proteins, which reversibly link the cortical actin cytoskeleton to the plasma membrane. Here, we describe a novel mouse model with global expression of moesinR171W that recapitulates multiple facets of patient disease, including severe lymphopenia. Further analysis reveals that these mice have diminished numbers of thymocytes and bone marrow precursors. X-MAID mice also exhibit systemic inflammation that is ameliorated by elimination of mature lymphocytes through breeding to a Rag1-deficient background. The few T cells in the periphery of X-MAID mice are highly activated and have mostly lost moesinR171W expression. In contrast, single-positive (SP) thymocytes do not appear activated and retain high expression levels of moesinR171W. Analysis of ex vivo CD4 SP thymocytes reveals defects in chemotactic responses and reduced migration on integrin ligands. While chemokine signaling appears intact, CD4 SP thymocytes from X-MAID mice are unable to polarize and rearrange cytoskeletal elements. This mouse model will be a valuable tool for teasing apart the complexity of the immunodeficiency caused by moesinR171W, and will provide new insights into how the actin cortex regulates lymphocyte function.

PD-1 and TIGIT Are Highly Co-Expressed on CD8+ T Cells in AML Patient Bone Marrow.

Despite the great success of immune-checkpoint inhibitor (ICI) treatment for multiple cancers, evidence for the clinical use of ICIs in acute myeloid leukemia (AML) remains inadequate. Further exploration of the causes of immune evasion in the bone marrow (BM) environment, the primary leukemia site, and peripheral blood (PB) and understanding how T cells are affected by AML induction chemotherapy or the influence of age may help to select patients who may benefit from ICI treatment. In this study, we comprehensively compared the distribution of PD-1 and TIGIT, two of the most well-studied IC proteins, in PB and BM T cells from AML patients at the stages of initial diagnosis, complete remission (CR), and relapse-refractory (R/R) disease after chemotherapy. Our results show that PD-1 was generally expressed higher in PB and BM T cells from de novo (DN) and R/R patients, while it was partially recovered in CR patients. The expression of TIGIT was increased in the BM of CD8+ T cells from DN and R/R patients, but it did not recover with CR. In addition, according to age correlation analysis, we found that elderly AML patients possess an even higher percentage of PD-1 and TIGIT single-positive CD8+ T cells in PB and BM, which indicate greater impairment of T cell function in elderly patients. In addition, we found that both DN and R/R patients accumulate a higher frequency of PD-1+ and TIGIT+ CD8+ T cells in BM than in corresponding PB, indicating that a more immunosuppressive microenvironment in leukemia BM may promote disease progression. Collectively, our study may help guide the combined use of anti-PD-1 and anti-TIGIT antibodies for treating elderly AML patients and pave the way for the exploration of strategies for reviving the immunosuppressive BM microenvironment to improve the survival of AML patients.

BTLA signaling is needed in newly generated T cells to block autoimmunity

Abstract Radiotherapy and/or chemotherapy in cancer treatment causes lymphopenia, which favors autoimmunity. Also, coinhibitory receptor blockade triggers immune-related adverse events in more patients than it cures of cancer. Having shown that PD-1 coinhibitory receptor is required in newly generated T cells (NGTC) to prevent lymphopenia-induced autoimmunity, our objective was to determine if the coinhibitory receptor BTLA is also required to establish tolerance in NGTC during lymphopenia. NGTC from thymocytes or established T cells from splenocytes of wildtype (WT) or BTLA−/− B6 mice were adoptively transferred to lymphopenic Rag−/− B6 mice. Sorted WT or BTLA−/− CD8 or CD4 single-positive (SP) thymocytes were adoptively transferred to Rag−/− mice not expressing MHC II (CiiTA−/−) or lacking MHC I (KbDb−/−), respectively. Disease was assessed by macroscopic appearance and weight loss. About 65% (5/8) recipients of BTLA−/− NGTC developed overt autoimmunity, while 4/4 recipients of BTLA−/− splenocytes were disease-free. 10/10 recipients of BTLA−/− CD8 SP T cells showed progressive weight gain. In contrast, 11/11 BTLA−/− CD4 SP T cell recipients had diarrhea, piloerection, hunched appearance, and progressive weight loss from 3–8 weeks post thymocyte transfer (p<0.0001). 3/11 BTLA−/− CD4 SP T cell recipients developed dermatitis post thymocyte transfer. Overt autoimmunity was evident in all WT Rag−/− (n=11) and Rag−/− KbDb−/− (n=4) recipients of thymocytes, but not in any of the CiiTA−/− recipients (n=4). There were no signs of disease in the recipients of WT NGTC. Our study suggests that BTLA is required to establish tolerance in NGTC. Disease in BTLA−/− thymocyte recipients was mediated by the CD4+ T cells and was dependent on MHC II.

OMIP 073: Analysis of human thymocyte development witha14-color flow cytometry panel.

This panel was designed for the identification and detailed characterization of the different developmental steps of human thymocytes. We optimized the panel for fresh tissue in order to provide an unbiased analysis of T cell development. Accurate selection of antibodies and precise gating allow us to phenotype 14 major stages of human thymocyte development and illustrate the trajectories of T cell development from early thymic progenitors (ETP) to mature T cells that are ready to populate the periphery. The panel identifies ETPs, T-lineage-committed cells (TC), CD34-positive immature single-positive CD4 cells (ISP4 CD34+), CD34-negative immature single-positive CD4 cells (ISP4 CD34-), CD45-low early double-positive cells (EDP CD45low), CD45-high early double-positive cells (EDP CD45high), late double-positive cells (LDP), single-positive CD4 cells (SP4), single-positive CD8 cells (SP8), ready-to-egress single-positive CD4 cells (rSP4), ready-to-egress single-positive CD8 cells (rSP8), T γδ cells (Tγδ), T regulatory cells (Treg), and ready-to-egress T regulatory cells (rTreg). To highlight important checkpoints during T cell development, we added antibodies relevant for specific developmental steps to the panel. These include CD1a to define TCs, CD28 as a marker for ß-selection and CD69 in combination with CD45RA to determine the maturation stage of thymocytes shortly before they become ready to egress the thymus and colonize the periphery. Moreover, Annexin V, as a marker for apoptosis, provides valuable extra information concerning the apoptotic death of thymocytes. Currently, we use this panel to identify aberrations in T cell development in health and disease.

Abnormal thymic B cell activation and impaired T cell differentiation in pristane-induced lupus mice

Changes in the thymus and potential mechanisms underlying the pathogenesis in pristane-induced lupus (PIL) mice are poorly understood. This study aimed to systematically and specifically examine changes in the thymus and the potential mechanisms responsible for immunological abnormalities in PIL mice. The results showed that PIL mice exhibit serious thymic hyperplasia, an elevated thymus index, a damaged histopathological structure and increased thymocyte apoptosis. We found that thymic T cell differentiation was impaired as the CD4+ CD8+ double-positive (DP) thymocyte frequency significantly decreased, becoming almost absent at 28 weeks after induction, while CD4 CD8- double-negative (DN) thymocytes and CD4+ CD8- single-positive (CD4+ SP) and CD4 CD8+ single-positive (CD8+ SP) cells were increased. This phenomenon might be explained by an inhibition of the DN-to-DP-cell transition and stimulation of DP cell conversion into CD4+ /CD8+ SP thymocytes. Moreover, we discovered a dramatic and abnormal increase in thymic B cells, that was associated with CD19, Irf8, Ebf1, Pax5, Irf4, Blk, CXCL13, CXCR5, CD79a, CD79b, Lyn, Syk, Btk, and BLNK gene accumulation, which exhibited positive interactions. We further verified that the mRNA expression of these genes was significantly upregulated and consistent with the RNA-seq results. These results suggest a role of these genes in the increase of B cells in the thymus of PIL mice. In summary, our results showed the changes in the thymus in PIL and elucidated the immunologic abnormalities of increased B cells, potentially providing insight into the associated molecular mechanisms and facilitating further research.

Deletion of Nemo-like Kinase in T Cells Reduces Single-Positive CD8+ Thymocyte Population.

The β-catenin/Wnt signaling pathway plays an important role in all stages of T cell development. Nemo-like kinase (NLK) is an evolutionary conserved serine/threonine kinase and a negative regulator of the Wnt signaling pathway. NLK can directly phosphorylate histone deacetylase 1 (HDAC1), as well as T cell factor/lymphoid enhancer-binding factor (TCF/LEF), causing subsequent repression of target gene transcription. By engineering mice lacking NLK in early stages of T cell development, we set out to characterize the role NLK plays in T cell development and found that deletion of NLK does not affect mouse health or lymphoid tissue development. Instead, these mice harbored a reduced number of single-positive (SP) CD8+ thymocytes without any defects in the SP CD4+ thymocyte population. The decrease in SP CD8+ thymocytes was not caused by a block in differentiation from double-positive CD4+CD8+ cells. Neither TCR signaling nor activation was altered in the absence of NLK. Instead, we observed a significant increase in cell death and reduced phosphorylation of LEF1 as well as HDAC1 among NLK-deleted SP CD8+ cells. Thus, NLK seems to play an important role in the survival of CD8+ thymocytes. Our data provide evidence for a new function for NLK with regard to its involvement in T cell development and supporting survival of SP CD8+ thymocytes.

Development, phenotypes of immune cells in BTBR T+Itpr3tf/J mice

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition that is characterized by a lack of social interaction, decreased verbal and non-verbal communication skills, and stereotyped repetitive behavior. There is strong evidence that a dysregulated immune response may influence neurodevelopment and thus may have a role in the development of ASD. This study focuses on the characterization of immune cell phenotypes in the BTBR T+Itpr3tf/J (BTBR) mouse strain, a widely used animal model for autism research. Our study demonstrated that BTBR mice have a different immune profile compared to C57BL/6J (B6) mice, which do not display ASD-like characteristics. Thymic cells of BTBR mice have more single positive (SP) CD4+ and CD8+ T cells and fewer double positive (DP) T cells than B6 mice. The development of T cells is increased in BTBR mice with regard to the double negative (DN4) population being much higher in BTBR mice. The spleens and blood of BTBR mice also have more T helper type 1 (Th1), T helper type 2 (Th2) and T regulatory (Treg) cells compared to B6 mice. Aire expression in the thymus and spleen of BTBR mice compared to B6 mice was equivalent and lower, respectively. The mature natural killer (NK) innate immune cell population in blood and spleen is lower in BTBR than B6 mice; NK cell development is blocked prior to the double positive (DN) CD11b+CD27+ stage in BTBR mice. Since BTBR mice have more CD4+ T cells and elevated numbers of Th1 (T-bet+) and Th2 (GATA3+) cells, their low defense against pathogen may be explained by the lower number of NK cells and the significantly lower Th1 to Th2 ratio. The elevated number of plasma cells and autoantibodies of BTBR mice may be due to less presence and function of splenic AIRE.

Transcriptome profiling of human thymic CD4+ and CD8+ T cells compared to primary peripheral T cells

BackgroundThe thymus is a highly specialized organ of the immune system where T cell precursors develop and differentiate into self-tolerant CD4+ or CD8+ T cells. No studies to date have investigated how the human transcriptome profiles differ, between T cells still residing in the thymus and T cells in the periphery.ResultsWe have performed high-throughput RNA sequencing to characterize the transcriptomes of primary single positive (SP) CD4+ and CD8+ T cells from infant thymic tissue, as well as primary CD4+ and CD8+ T cells from infant and adult peripheral blood, to enable the comparisons across tissues and ages. In addition, we have assessed the expression of candidate genes related to autoimmune diseases in thymic CD4+ and CD8+ T cells. The thymic T cells showed the largest number of uniquely expressed genes, suggesting a more diverse transcription in thymic T cells. Comparing T cells of thymic and blood origin, revealed more differentially expressed genes, than between infant and adult blood. Functional enrichment analysis revealed an over-representation of genes involved in cell cycle and replication in thymic T cells, whereas infant blood T cells were dominated by immune related terms. Comparing adult and infant blood T cells, the former was enriched for inflammatory response, cytokine production and biological adhesion, while upregulated genes in infant blood T cells were associated with cell cycle, cell death and gene expression.ConclusionThis study provides valuable insight into the transcriptomes of the human primary SP T cells still residing within the thymus, and offers a unique comparison to primary blood derived T cells. Interestingly, the majority of autoimmune disease associated genes were expressed in one or more T cell subset, however ~ 11% of these were not expressed in frequently studied adult peripheral blood.

The aged thymus is impaired in supporting T cell central tolerance

Abstract During aging, the thymus undergoes involution, decreasing dramatically in size and mass, and becoming increasingly disorganized. Notably, thymic epithelial cells decline with age, especially within the compartment of mature medullary thymic epithelial cells (mTECs) that express and present tissue-restricted antigens to thymocytes. Age-associated changes in the composition of hematopoietic antigen-presenting cells (APCs) have also been observed, but are not well characterized. Given that both mTECs and APCs present diverse self-antigens to thymocytes, we hypothesized that central tolerance is impaired in the aged thymus. Using live-cell two-photon microscopy, we determined that CD4-single positive (CD4SP) thymocytes, regardless of thymocyte age, had reduced motility and accumulation within the medulla of an aged thymus. We report age-associated changes in both the localization and composition of thymic APC subsets. To test whether this altered thymic environment functionally impacted central tolerance, we used thymic slice assays to measure tolerance induction of CD8− and CD4SP thymocytes encountering a range of model self-antigens. These assays indicated that central tolerance in the aged thymus was impaired, particularly against low-avidity auto-antigens. Our findings indicate that newly generated T cells selected in an aged thymic environment have not undergone a rigorous screening process against autoimmunity.

Transcriptomic features of tumour-infiltrating CD4lowCD8high double positive \u03b1\u03b2 T cells in melanoma

Peripheral CD4+CD8+ double positive (DP) T cells are a phenotypically and functionally heterogeneous population depending on their origin and pathologic context. We previously identified among tumour infiltrating lymphocytes in melanoma, a tumour-reactive MHC class-I restricted CD4lowCD8high DP αβ T-cell subpopulation with CD4-like function. In this study, we used an in-depth comparative transriptomic analysis of intra-melanoma DP T cells and CD4 and CD8 single positive (SP) T cells, to better comprehend the origin of this DP phenotype, and define the transcriptomic signature of activated DP T cells. We observed that intra-melanoma DP T cells were transcriptome-wise closer to their CD8 SP T-cell counterparts in terms of number of genes differentially expressed (97 in common with CD8 SP T cells and 15 with CD4 SP T cells) but presented hallmarks of a transition to a CD4-like functional profile (CD40LG) with a decreased cytotoxic signature (KLRC1) in favour of an increased cytokine-receptor interaction signature (IL4, IL24, IL17A…). This unleashed CD4-like program could be the results of the observed unbalanced expression of the THPOK/Runx3 transcription factors in DP T cells. Overall, this study allow us to speculate that intra-melanoma DP T cells arise from CD8 SP T cells being reprogrammed to a helper function.

Quantitative, qualitative and spatial analysis of lymphocyte infiltration in periampullary and pancreatic adenocarcinoma.

Immunotherapeutic modalities are currently revolutionizing cancer treatment. In pancreatic cancer, however, early clinical trials have been disappointing. The optimization of immunotherapeutic strategies requires better understanding of the inflammatory tumor microenvironment. Therefore, the aim of our study was to perform a detailed in situ description of lymphocyte infiltration patterns in resected pancreatic and other periampullary cancers. Multiplexed immunofluorescence imaging was applied to tissue microarrays with tumors from a cohort of 175 patients with resected periampullary adenocarcinoma. A panel of immune cell markers including CD4, CD8α, FoxP3, CD20, CD45RO and pan-cytokeratin was applied to allow for simultaneous spatial analysis of multiple lymphocyte populations. The majority of lymphocyte populations were significantly more abundant in intestinal (I-type) compared to pancreatobiliary (PB-type) tumors. Hierarchical cluster analysis revealed several immune cell signatures of potential clinical relevance. Notably, in the stromal compartment of PB-type tumors, high infiltration of B cells, CD8α+ CD45RO+ and single-positive CD4+ T cells, but low levels of FoxP3+ CD45ROhigh and single-positive CD8α+ T cells were associated with improved overall survival (OS). The study also defined prognostic relevant topographical patterns of lymphocytic infiltration, in particular proximity of CD8α+ cells to cancer cells. Moreover, the presence of lymphocytes with potential T-helper capacities (CD4+ ) in the nearest vicinity to CD8α+ cells was associated with a prolonged OS. Our data demonstrate that the composition and clinical impact of immune infiltrates in periampullary adenocarcinoma differ by morphological type as well as localization. Furthermore, spatial in situ analysis identified potential immunological mechanisms of prognostic significance.

Intra-uterine growth restriction induced by maternal low-protein diet causes long-term alterations of thymic structure and function in adult male rat offspring.

Early malnutrition, the first environmental cause of intra-uterine growth restriction, impairs development of the thymus. Alterations of the thymic structure and function are reported at young ages in murine and ovine models. However, descriptions of thymic consequences of fetal malnutrition at adulthood are scarce. The present study investigates thymic structure, protein expression and cell selection process observed at postnatal day 180 (PND180) in male offspring of rats exposed to maternal low-protein diet (mLPD) compared with control diet during gestation. The thymic index was lower in adult offspring exposed to mLPD (P<0·05). The thymic cortico-medullar ratio was lower in adult offspring exposed to mLPD (P<0·05). At PND180, the protein expression of the lymphotoxin β receptor (P<0·05), the autoimmune regulator (P<0·05) and Forkhead Box P3 (FoxP3; P<0·05) was all significantly lower in the mLPD group. The CD4+:CD8+ single-positive thymocyte subpopulation ratio and CD4+:CD8+ lymphocyte subpopulation ratio were increased in the mLPD group (P<0·05). Among CD3+ lymphocytes, the proportions of CD4+CD8+ double-positive lymphocytes, CD31+ recent thymic emigrants and CD4+FoxP3+ lymphocytes were not significantly different between mLPD and control groups. These findings suggest mLPD during gestation induced long-lasting alterations in the development of thymic structure and thymic cell maturation and selection process in adult male rat offspring.

Effects of yolkin on the immune response of mice and its plausible mechanism of action

Yolkin is a product of proteolytic degradation of vitellogenin, a protein contained in eggs’ yolk, with already described procognitive properties. Here, we investigated effects of yolkin on the humoral and cellular immune response in mice, phenotype of cells from lymphoid organs and function of innate immunity cells. In vitro studies included effects of yolkin on mitogen-induced thymocyte proliferation, percentage of CD19 cells in bone marrow cells culture, expression of signaling molecules in Jurkat cells, interleukin 2 receptor (IL-2R) subunits in WEHI 231 cells and susceptibility of these cells to anti-Ig-induced cell death. The results showed that repeatable i.p. injections of yolkin stimulated the humoral immune response to sheep red blood cells (SRBC) irrespective of the time of the treatment. On the other hand, yolkin inhibited contact sensitivity to oxazolone. Treatment of mice with yolkin diminished the percentage of double positive cells and increasing the content of single positive CD4+ and CD8+ cells in the thymus. At the same time an increase of percentage of CD19 + B cells in the spleen and mesenteric lymph nodes was observed. In addition, the protein, given i.p., diminished ex vivo ability to synthesize nitric oxide by resident, peritoneal macrophages, stimulated with lipopolisaccharide (LPS). In vitro studies showed that yolkin increased CD19+ cell content in bone marrow cell population. The protein also enhanced proliferation of thymocytes to concanavalin A and stimulated expression of MAP kinases in Jurkat cells. In WEHI 231 B cell line yolkin caused a loss of IL-2R gamma chain expression, correlated with an increased resistance of these cells to proapoptotic action of anti-Ig antibodies. In conclusion, this is a first demonstration of immunotropic properties of yolkin in in vitro and in vivo tests. The results provide evidence for induction of maturation and stimulatory signals in immature T and B cells by the protein, suggesting its potential role in the development of an embryo’s immune system.

Adhesion between medullary thymic epithelial cells and thymocytes is regulated by miR-181b-5p and miR-30b*

In this work, we demonstrate that adhesion between medullary thymic epithelial cells (mTECs) and thymocytes is controlled by miRNAs. Adhesion between mTECs and developing thymocytes is essential for triggering negative selection (NS) of autoreactive thymocytes that occurs in the thymus. Immune recognition is mediated by the MHC / TCR receptor, whereas adhesion molecules hold cell-cell interaction stability. Indeed, these processes must be finely controlled, if it is not, it may lead to aggressive autoimmunity. Conversely, the precise molecular genetic control of mTEC-thymocyte adhesion is largely unclear. Here, we asked whether miRNAs would be controlling this process through the posttranscriptional regulation of mRNAs that encode adhesion molecules. For this, we used small interfering RNA to knockdown (KD) Dicer mRNA in vitro in a murine mTEC line. A functional assay with fresh murine thymocytes co-cultured with mTECs showed that single-positive (SP) CD4 and CD8 thymocyte adhesion was increased after Dicer KD and most adherent subtype was CD8 SP cells. Analysis of broad mTEC transcriptional expression showed that Dicer KD led to the modulation of 114 miRNAs and 422 mRNAs, including those encoding cell adhesion or extracellular matrix proteins, such as Lgals9, Lgals3pb, Tnc and Cd47. Analysis of miRNA-mRNA networks followed by miRNA mimic transfection showed that these mRNAs are under the control of miR-181b-5p and miR-30b*, which may ultimately control mTEC-thymocyte adhesion. The expression of CD80 surface marker in mTECs was increased after Dicer KD following thymocyte adhesion. This indicates the existence of new mechanisms in mTECs that involve the synergistic action of thymocyte adhesion and regulatory miRNAs.

IL-2 production by self-reactive CD4 thymocytes scales regulatory T cell generation in the thymus.

Regulatory T (T reg) cells, a specialized subset of CD4+ T cells, are essential to prevent fatal autoimmunity. Expression of the T reg lineage-defining transcription factor Foxp3, and therefore their differentiation in the thymus, is dependent upon T cell receptor (TCR) and interleukin-2 (IL-2) signaling. Here, we report that the majority of IL-2-producing cells in the thymus are mature CD4 single-positive (CD4SP) thymocytes and that continuous IL-2 production sustained thymic T reg cell generation and control of systemic immune activation. Furthermore, single-cell RNA sequencing analysis of CD4 thymocyte subsets revealed that IL-2 was expressed in self-reactive CD4SP thymocytes, which also contain T reg precursor cells. Thus, our results suggest that the thymic T reg cell pool size is scaled by a key niche factor, IL-2, produced by self-reactive CD4SP thymocytes. This IL-2-dependent scaling of thymic T reg cell generation by overall self-reactivity of a mature post-selection thymic precursor pool may likely ensure adequate control of autoimmunity.

Thymic-specific regulation of TCR signaling by Tespa1.

Double-positive (DP) thymocytes undergo positive selection to become mature single-positive CD4+ and CD8+ T cells in response to T cell receptor (TCR) signaling. Unlike mature T cells, DP cells must respond to low-affinity self-peptide-MHC ligands before full upregulation of their surface TCR expression can occur. Thus, DP thymocytes must be more sensitive to ligands than mature T cells. A number of molecules have been found that are able to enhance the strength of the TCR signal to facilitate positive selection. However, almost all of these molecules are also active in mature T cells. Themis (thymocyte expressed molecule involved in selection) and Tespa1 (thymocyte expressed positive selection associated 1) are two recently discovered molecules essential for optimal TCR signaling and thymocyte development. A deficiency in both molecules leads to defects in positive selection. Here, we compared the relative contributions of Themis and Tespa1 to positive selection in thymocytes. We show that Tespa1 deficiency led to more limited and specific gene expression profile changes in cells undergoing positive selection. In mixed bone marrow transfer experiments, Tespa1-/- cells showed more severe defects in thymocyte development than Themis-/- cells. However, Tespa1-/- cells showed a substantial degree of homeostatic expansion and became predominant in the peripheral lymphoid organs, suggesting that Tespa1 is a thymic-specific TCR signaling regulator. This hypothesis is further supported by our observations in Tespa1 conditional knockout mice, as Tespa1 deletion in peripheral T cells did not affect TCR signaling or cell proliferation. The different regulatory effects of Tespa1 and Themis are in accordance with their nonredundant roles in thymocyte selection, during which Tespa1 and Themis double knockouts showed additive defects.

Abstract 2342: Experimental modeling of acute- and chronic-GvHD by xenotransplanting human donor PBMCs or cord blood CD34+ cells (HSC) into NSG mice

Abstract Graft vs. host diseases (GvHD) accounts for 15-30% of deaths following allogenic hematopoietic stem cell transplantation (allo-HSCT) for treatment of malignant diseases. Acute GvHD (aGvHD) typically involves skin, gastrointestinal and hepatic inflammation, and occurs within 100 days of transplantation; Chronic GvHD (cGvHD) involves multiple organs and occurs beyond 100 days. aGvHD is largely due to the rapid activation of donor T cells (Th1, CD8+ biased), causing tissue damage and often leading to mortality; in contrast, cGvHD is Th2 biased, typically display autoimmune-like syndrome, involving both T- and B-cell. Currently, GvHD models are mostly allo-transplantations between mice, which are criticized for poor physiological relevance. Here, we present Xenotransplantation of human donor into NSG mice, which will address some of the limitation of current models. Human peripheral blood mononuclear cells (PBMC) derived from normal donors were transplanted into NSG or NSG-like strains for modeling aGvHD. Cord blood derived HSCs (hCD34+) NSG (Jackson) were transplanted for modeling cGvHD. Hosts were monitored twice weekly, including clinical observations (e.g. animal postures, activity, fur texture, and skin integrity), body weight changes, gross pathology and histopathology upon termination, along with human-immunological phenotyping of peripheral blood, spleen, lung, liver and skin by flow cytometry, histopathology and/or immunohistochemistry. In addition, GvHD novel biomarkers Elafin, IL-18, REG3α and ST2, measured by ELISA (MBL International) at baseline and post cells engraftment. NSG mice engrafted with human PBMC, or purified T-cells, from normal donors rapidly developed typical symptoms of aGvHD, as early as 4 weeks post transplantation, including severe body loss, reduced activity, hunched posture, loss of fur, severe ruffling and overall poor grooming. Significant engraftment of human CD45+ cells were detected up to 6 weeks post-engraftment dominated with CD3+ human T-cells of single positive CD4+ or CD8+ T-cells. The corresponding kinetics of clinical symptoms in parallel with the degree of engraftment of T cells suggests xenografting were responsible for aGvHD. As for the cGvHD, NSG-mice engrafted with CD34+ cells derived from cord blood with cGvHD high- risk HLA haplotypes C*0602 and C*0401. Manifestation of cGvHD was observed 18 weeks to 39 weeks post-engraftment; symptoms were similar to aGvHD, but also include facial/full body alopecia, and scaly skin. Interestingly, in correlation of this, human engraftment of CD45+, particularly T-cells including CD4+ and their CD30+ subsets, are kinetically increased in blood and spleen with similar timeline, suggesting their roles in the observed cGvHD. The xenograft murine model using adult human PBMC and cord blood derived CD34+ HSCs could be alternative experimental systems to model human aGvHD and cGvHD for investigating disease mechanisms and evaluating treatment strategy. Citation Format: Ann E. Lin, Annie X. An, Mingfa Zang, Derron Yu, Eunmi Hwang, Israel Romero, Linda Quirino, Marybeth George, Pirouz Daftarian, Wenqing Yang, Henry Li. Experimental modeling of acute- and chronic-GvHD by xenotransplanting human donor PBMCs or cord blood CD34+ cells (HSC) into NSG mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2342.

Oxaliplatin Treatment Alters Systemic Immune Responses.

Purpose Oxaliplatin is a platinum-based chemotherapeutic agent demonstrating significant antitumor efficacy. Unlike conventional anticancer agents which are immunosuppressive, oxaliplatin has the capacity to stimulate immunological effects in response to the presentation of damage associated molecular patterns (DAMPs) elicited upon cell death. However, the effects of oxaliplatin treatment on systemic immune responses remain largely unknown. Aims of this study were to investigate the effects of oxaliplatin treatment on the proportions of (1) splenic T cells, B cells, macrophages, pro-/anti-inflammatory cytokines, gene expression of splenic cytokines, chemokines, and mediators; (2) double-positive and single-positive CD4+ and CD8+ T thymocytes; (3) bone-marrow hematopoietic stem and progenitor cells. Methods Male BALB/c mice received intraperitoneal injections of oxaliplatin (3mg/kg/d) or sterile water tri-weekly for 2 weeks. Leukocyte populations within the spleen, thymus, and bone-marrow were assessed using flow cytometry. RT-PCR was performed to characterise changes in splenic inflammation-associated genes. Results Oxaliplatin treatment reduced spleen size and cellularity (CD45+ cells), increased the proportion of CD4+, CD8+, and Treg cells, and elevated TNF-α expression. Oxaliplatin was selectively cytotoxic to B cells but had no effect on splenic macrophages. Oxaliplatin treatment altered the gene expression of several cytokines, chemokines, and cell mediators. Oxaliplatin did not deplete double-positive thymocytes but increased the single-positive CD8+ subset. There was also an increase in activated (CD69+) CD8+ T cells. Bone-marrow hematopoietic progenitor pool was demonstrably normal following oxaliplatin treatment when compared to the vehicle-treated cohort. Conclusion Oxaliplatin does not cause systemic immunosuppression and, instead, has the capacity to induce beneficial antitumor immune responses.