T-cell Populations In Patients Research Articles

Janus Kinase (JAK) 1 Inhibition Results in Significant Changes in Serum Proteins and Peripheral T-Cell Populations That Correlated with Clinical Scores in Chronic Graft Versus Host Disease (GVHD) Patients (an Analysis from GRAVITAS-309)

Background: Chronic GVHD (cGVHD) occurs in 30-70% of allogeneic hematopoietic cell transplantation patients, and the rate has increased over the past 2 decades. Based on the degree of organ involvement, cGVHD is classified as mild, moderate, or severe. Standard treatment includes systemic corticosteroids (CS), together with immunosuppressive agents. GRAVITAS-309 (NCT03584516) was designed as a 2-part, multicenter, randomized trial to assess efficacy and safety of itacitinib, an orally bioavailable, selective JAK1 inhibitor, in combination with CS as first-line treatment for moderate to severe cGVHD. The dose-finding, open-label, randomized portion of the study initially investigated itacitinib at 200 mg once daily (qd) and 300 mg qd in combination with CS (methylprednisolone or prednisone); both doses were well tolerated. The trial was expanded to test itacitinib 400 mg qd and 300 mg twice daily (bid); the initial 300 mg qd cohort was expanded and a CS monotherapy cohort was added. Results from the expanded portion of the study (n=139 patients) showed improved overall response rate (ORR) at 6 months in patients treated with itacitinib + CS vs CS alone (Im, et al. Blood 2022;140[Suppl 1]:1870). Here, we analyzed samples collected from patients in 4 cohorts (cohort A [300 mg qd], cohort B [400 mg qd], cohort C [300 mg bid], and cohort D [CS alone]) for systemic proteins and immune cell populations, to support investigation into the itacitinib mechanism of action and to correlate with clinical outcomes. Methods: Peripheral blood mononuclear cells (PBMCs) and serum were collected before treatment (baseline, day 1), and day 7, day 14 (PBMCs only), and day 28 following treatment initiation. Serum proteins were analyzed using the OLINK Target 96 platform, and circulating T-cell subsets were measured using flow cytometry of PBMCs and presented as a percentage of parent population. Samples were assessed irrespective of patients' response. Most of the patients (70-100% per cohort) with samples available for analysis were responders (achieved complete or partial responses). Results: Serum levels of proteins, including several proposed as potential prognostic cGVHD biomarkers, changed significantly at day 28 relative to baseline following treatment with itacitinib (Table 1). Levels of elafin, a marker of acute GVHD of the skin, were significantly reduced and levels of dickkopf-3 (DKK3), a marker of sclerotic and nonsclerotic cGVHD, were significantly increased in itacitinib cohorts only. Based on a repeated measures correlation analysis, both markers significantly correlated with overall clinical skin scores (elafin: r=+0.43; DKK3: r=-0.53; both P<0.001). Serum levels of other reported markers of cGVHD, including osteopontin, C-X-C motif chemokine ligand (CXCL) 9 and CXCL10, were significantly decreased exclusively in itacitinib-treated cohorts, and not in the CS only cohort. Some proteins, including matrix metalloproteinase (MMP) 3, increased in all cohorts, suggesting that changes in these proteins cannot be attributed to itacitinib alone, but may reflect response to CS; other proteins, such as suppression of tumorigenicity (ST2), remained unchanged in response to treatment in all cohorts. CS treatment alone resulted in an increase in naive CD4 T cells (CD4 +CD8 ->CD45RA +CCR7 -) and a corresponding reduction of CD4 effector memory T cells (CD4 +CD8 ->CD45RA -CCR7 +) within 28 days (Figure 1, cohort D). Changes in naive CD4 T cells following CS monotherapy were significantly attenuated by itacitinib ( P<0.05 vs CS at day 28), particularly at the 400 mg qd dose (Figure 1, cohort B), which demonstrated the best ORR at 6 months (53% with 400 mg qd vs 36% with CS). Conclusions: Analysis of patient samples from GRAVITAS-309 revealed that serum elafin and DKK3 levels significantly correlated with skin GVHD scores, possibly reflecting the systemic origin of observed skin manifestations. Serum levels of osteopontin, CXCL9, and CXCL10 were specifically reduced by itacitinib in patients with cGVHD and may represent potential pharmacodynamic markers. Observed changes in different peripheral T-cell populations in patients' samples following itacitinib treatment were consistent with those reported in murine GVHD models, suggesting a potential role of naive CD4 + T cells in driving this disease. Planned future analyses will include identification of novel biomarkers that correlate with organ involvement.

Tissue-Resident Memory T Cells in Pancreatic Ductal Adenocarcinoma Coexpress PD-1 and TIGIT and Functional Inhibition Is Reversible by Dual Antibody Blockade.

Pancreatic ductal adenocarcinoma (PDAC) has a poor clinical outlook. Responses to immune checkpoint blockade are suboptimal and a much more detailed understanding of the tumor immune microenvironment is needed if this situation is to be improved. Here, we characterized tumor-infiltrating T-cell populations in patients with PDAC using cytometry by time of flight (CyTOF) and single-cell RNA sequencing. T cells were the predominant immune cell subset observed within tumors. Over 30% of CD4+ T cells expressed a CCR6+CD161+ Th17 phenotype and 17% displayed an activated regulatory T-cell profile. Large populations of CD8+ tissue-resident memory (TRM) T cells were also present and expressed high levels of programmed cell death protein 1 (PD-1) and TIGIT. A population of putative tumor-reactive CD103+CD39+ T cells was also observed within the CD8+ tumor-infiltrating lymphocytes population. The expression of PD-1 ligands was limited largely to hemopoietic cells whilst TIGIT ligands were expressed widely within the tumor microenvironment. Programmed death-ligand 1 and CD155 were expressed within the T-cell area of ectopic lymphoid structures and colocalized with PD-1+TIGIT+ CD8+ T cells. Combinatorial anti-PD-1 and TIGIT blockade enhanced IFNγ secretion and proliferation of T cells in the presence of PD-1 and TIGIT ligands. As such, we showed that the PDAC microenvironment is characterized by the presence of substantial populations of TRM cells with an exhausted PD-1+TIGIT+ phenotype where dual checkpoint receptor blockade represents a promising avenue for future immunotherapy.

Poster 125: Determining Biological Age and Prolonging the Healthspan by Targeting Senescence

Poster 125: Determining Biological Age and Prolonging the Healthspan by Targeting Senescence

The mitochondrial DNA constitution shaping T-cell immunity in patients with rectal cancer at high risk of metastatic progression

PurposeA significant percentage of colorectal cancer patients proceeds to metastatic disease. We hypothesised that mitochondrial DNA (mtDNA) polymorphisms, generated by the high mtDNA mutation rate of energy-demanding clonal immune cell expansions and assessable in peripheral blood, reflect how efficiently systemic immunity impedes metastasis.Patients and methodsWe studied 44 rectal cancer patients from a population-based prospective biomarker study, given curative-intent neoadjuvant radiation and radical surgery for high-risk tumour stage and followed for metastatic failure. Blood specimens were sampled at the time of diagnosis and analysed for the full-length mtDNA sequence, composition of immune cell subpopulations and damaged serum mtDNA.ResultsWhole blood total mtDNA variant number above the median value for the study cohort, coexisting with an mtDNA non-H haplogroup, was representative for the mtDNA of circulating immune cells and associated with low risk of a metastatic event. Abundant mtDNA variants correlated with proliferating helper T cells and cytotoxic effector T cells in the circulation. Patients without metastatic progression had high relative levels of circulating tumour-targeting effector T cells and, of note, the naïve (LAG-3+) helper T-cell population, with the proportion of LAG-3+ cells inversely correlating with cell-free damaged mtDNA in serum known to cause antagonising inflammation.ConclusionNumerous mtDNA polymorphisms in peripheral blood reflected clonal expansion of circulating helper and cytotoxic T-cell populations in patients without metastatic failure. The statistical associations suggested that patient’s constitutional mtDNA manifests the helper T-cell capacity to mount immunity that controls metastatic susceptibility.Trial registrationClinicalTrials.gov NCT01816607; registration date: 22 March 2013.

Exploratory study on immune phenotypes in Alzheimer’s disease and vascular dementia

The differentiation of Alzheimer's disease (AD) dementia from vascular dementia (VaD) and mixed-type dementia (mixed dementia) requires stepwise analysis and usually occurs late in the disease process. Early diagnosis and therapy monitoring would benefit greatly from the identification of biomarkers of neurodegeneration, especially blood biomarkers. To this end, the aim of the present pilot study was to investigate differences in the distribution of peripheral T-cell populations in patients with AD compared to VaD and mixed dementia. Flow cytometry was performed on blood samples from 11 patients with AD, six with VaD and six with mixed dementia, as well as 17 healthy control subjects (HCs). CD4+ and CD8+ T cells were typed for expression of CD45, CD27, CD28, CD25, FoxP3, CCR4 and CCR6; the other leukocytes were also assessed. Functionally, immune cell uptake of the β-amyloid (Aβ) toxic fragment (Aβ1-42 ) was also evaluated. A higher proportion of CD4+CD28- memory T cells and a reciprocal reduction of CD4+CD28+CD27+ naïve T lymphocytes was detected in all patient groups relative to controls. Significantly fewer CD4+CD25+FoxP3 regulatory T cells were present in patients with VaD, and significantly more CCR6+ and CCR4+ CD4+ T cells in those with AD. Higher CCR6+ T-cell frequencies were also present in patients with mixed dementia, potentially due to the inflammation and immune cell chemoattraction triggered by Aβ. The present study was a comprehensive investigation comparing different kinds of dementia, revealing differentially expressed peripheral markers that are potentially useful for early AD, VaD and mixed dementia diagnoses, and that would assist in proper treatments for these disparate diseases. Validation is now required.

Abstract PR3: Development of a transcriptomic T-cell atlas highlights the differential role of T-cell subpopulations in CAR T-cell therapy resistance

Abstract Chimeric antigen receptor (CAR) T-cell therapy has been a major breakthrough in B-cell acute lymphoblastic leukemia (B-ALL), yet therapeutic resistance remains an important clinical challenge. Given that approximately 20% of patients do not respond to therapy and 40% of initial responders relapse within a year, there is a pressing need to understand the basis of therapy resistance in order to identify prognostic biomarkers and improve the therapeutic modality. We hypothesize that intrinsic T-cell factors are a major contributor to early therapeutic failure to CAR T-cell therapy. Most protocols do not select for specific T-cell subtypes, leading to the use of heterogeneous T-cell populations for CAR T-cell engineering. In order to characterize the molecular phenotypes of T cells underlying therapeutic response, we performed subtype-specific transcriptomic profiling of clinical T-cell populations in patients subsequently treated with anti-CD19 CAR T-cell therapy. We identified 71 patients with B-ALL treated with CAR T-cell therapy at the Children’s Hospital of Philadelphia, and obtained T cells via clinical leukapheresis prior to CAR T-cell engineering. We used FACS to separate T cells into five subtypes per patient (Naive, Stem Cell Memory, Central Memory, Effector Memory, and Effector), and performed bulk RNA-sequencing on all 355 samples. To our knowledge, this is the largest and most comprehensive transcriptomic profiling of clinical T cells in CAR T-cell therapy to date. We find that a higher proportion of CCR7+CD62L+ naive and early memory T-cell phenotypes associates with longer clinical T-cell persistence (HR=3.25 [1.37-7.89], p=0.006), and that combining proportions of these populations with CCR7−CD62L−CD45RO+CD95+ effector memory T cells has the greatest prognostic value (HR: 4.82 [1.89-12.32]; p<0.001). We identified global transcriptomic markers of early and differentiated T-cell populations and identified differentially expressed genes that discriminated between clinical responders and nonresponders. Single-sample gene set enrichment analysis identified an enrichment of T-cell exhaustion pathways among the clinical nonresponders, which was predominant in the early lineage T-cell phenotypes (p=0.018-0.095) but not in the effector T-cell phenotypes (p=0.15-0.37). Together, these data shed light on the critical role of preinfusion T-cell populations in CAR T-cell therapy, and may inform clinical prognosis and the development of improved CAR T-cell therapies. This abstract is also being presented as Poster A65. Citation Format: Gregory M. Chen, Changya Chen, Rajat K. Das, Yang-Yang Ding, Bing He, Hannah Kim, David M. Barrett, Kai Tan. Development of a transcriptomic T-cell atlas highlights the differential role of T-cell subpopulations in CAR T-cell therapy resistance [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr PR3.

γδ T-cell function is inhibited in end-stage renal disease and impacted by latent tuberculosis infection

Patients with end-stage renal disease (ESRD) are at elevated risk of acquiring infectious diseases, including tuberculosis (TB). Inflammation and uremia negatively impact immune function in this population, but specific pathways involved in TB immunity have not been identified. Although γδ T cells are known to contribute to protection from TB, their phenotype and function in patients with ESRD is relatively unknown. To determine this we recruited 20 patients with and 20 without ESRD (controls), with or without latent TB infection to assess γδ T cell frequency, surface phenotype, and cytokine production by flow cytometry in response to stimulation. γδ T cells derived from patients with ESRD exhibited significantly lower expression of CCR5, CXCR3, and CD26 compared to controls. Furthermore, patients with ESRD, particularly the group with latent TB infection, exhibited poor IFNγ, TNFα, and GMCSF responses to stimulation with either phosphoantigen HMB-PP, IL-12/IL-18, E.coli, or phorbol myristate acetate and ionomycin. Similar dysfunctional responses were observed in patients with active TB. Surprisingly, neither the γδ phenotype nor its function was associated with plasma markers of inflammation or microbial translocation. Thus, there is significant perturbation of the γδ T-cell population in patients with ESRD, particularly in those with latent TB infection.

Increase of circulating α4β7+ conventional memory CD4 and regulatory T cells in patients with common variable immunodeficiency (CVID)

This study investigated whether circulating α4β7+ expressing T cells could serve as a potential marker for gastrointestinal (GI) disease activity in patients with CVID. The analysis of α4β7+ T cells in the peripheral blood of 36 patients and 22 healthy donors (HD) revealed increased percentages of α4β7+ conventional memory CD4 T cells and Tregs, but not among CD8 T-cell populations in patients with CVID compared to HD. No differences between patients with and without chronic or acute GI symptoms were observed. EUROClass smB− and 21lo patients, had higher percentages of α4β7+ memory CD4 T cells compared to HD and smB+ or 21norm patients, respectively. In summary, the detection of α4β7+ T cells in the peripheral blood did not correlate with active or chronic gastrointestinal disease. The increase of these cells in smB− and 21lo patients adds another piece to the immune dysregulation observed in these patients.

CD73 is a phenotypic marker of effector memory Th17 cells in inflammatory bowel disease

Purinergic signaling and associated ectonucleotidases, such as CD39 and CD73, have been implicated in the pathogenesis of inflammatory bowel disease (IBD). CD39 is known to be a Treg memory cell marker, and here we determine the phenotype and function of CD73(+) CD4(+) T lymphocytes in patients with IBD. We describe elevated levels of CD73(+) CD4(+) T cells in the peripheral blood and intestinal lamina propria of patients with active IBD. The functional phenotype of these CD73(+) CD4(+) T cells was further determined by gene expression, ecto-enzymatic activity, and suppressive assays. Increased numbers of CD73(+) CD4(+) T cells in the periphery and lamina propria were noted during active inflammation, which returned to baseline levels following anti-TNF treatment. Peripheral CD73(+) CD4(+) T cells predominantly expressed CD45RO, and were enriched with IL-17A(+) cells. The CD73(+) CD4(+) cell population expressed higher levels of RORC, IL-17A, and TNF, and lower levels of FOXP3 and/or CD25, than CD73(-) CD4(+) T cells. Expression of CD73 by peripheral CD4(+) T cells was increased by TNF, and decreased by an anti-TNF monoclonal antibody (infliximab). In vitro, these peripheral CD73(+) CD4(+) T cells did not suppress proliferation of CD25(-) effector cells, and expressed higher levels of pro-inflammatory markers. We conclude that the CD73(+) CD4(+) T-cell population in patients with active IBD are enriched with cells with a T-helper type 17 phenotype, and could be used to monitor disease activity during treatment.

Treg Lymphocytes in Autoimmune Uveitis

Purpose: To evaluate circulating CD4+CD25+ regulatory T-cell populations in patients with autoimmune uveitis and to assess whether T-regulatory cell populations correlate with clinical features.Methods: Sixty-four patients with noninfectious uveitis were enrolled. Following isolation and purification of peripheral blood mononuclear cells, Tregs were analyzed by assessing FOXP3 expression on T cells with an APC anti-human FOXP3 staining kit. Samples were simultaneously stained with anti-CD25-PeCy7, anti-CD3-FITC, anti-CD4-PercP, and anti-CD152 (CTLA-4)-PE monoclonal antibodies.Results: A decrease in CD4+CD25+FOXP3+ T cells was detected in patients with active uveitis compared with healthy controls (p < .05). In patients achieving clinical remission, the percentage of CD4+CD25+FOXP3+ T cells increased up to values comparable to those of healthy controls, in step with an increased expression of CTLA4.Conclusion: Therapy of autoimmune uveitis results in the increase of T-regulatory lymphocyte population and restoration of their functional state. These changes are likely to contribute to the patients' clinical improvement or disease regression.

HIV infection perturbs interleukin-7 signaling at the step of STAT5 nuclear relocalization

Interleukin-7 (IL-7) responses are impaired in CD4(+) T cells from HIV-infected patients, which may play a significant role in the loss of CD4(+) T-cell homeostasis. We set to investigate the nature of IL-7-dependent signaling defects in patients with progressive HIV-1 infection. IL-7 signaling was compared in CD4(+) T cells from viremic patients with a viral load more than 10,000 copies of HIV RNA/ml (n = 23) and from healthy blood donors (n = 23). Phosphorylation of the transcription factor STAT5 on the regulatory serine S726 and the key tyrosine Y694 was monitored by intracellular flow cytometry. Phospho-STAT5 relocalization to the nucleus was analyzed by quantitative immunofluorescence imaging. In control CD4(+) T cells, S726 phosphorylation was mostly constitutive and inducible by IL-7 to a limited extent (1.3x, P < 0.05). In contrast, phosphorylation at Y694 was highly inducible by IL-7 (12.6x, P < 0.0001). Progressive HIV infection led to hyperphosphorylation of both S726 and Y694 in naive CD4(+) T cells, with these changes correlating together (R = 0.66, P = 0.01). Quantitative image analysis revealed an impairment in the nuclear relocalization of both forms of phospho-STAT5 in patient cells (P < 0.005 for S726; P < 0.05 for Y694). The nuclear relocalization defect correlated with increased HLA-DR expression (R = 0.75, P < 0.01), suggesting a role for chronic immune activation in perturbed IL-7 signal transduction. HIV infection perturbs IL-7 signaling by impairing the access of STAT5 to the nuclear compartment. This defect may contribute to the loss of CD4(+) T-cell populations in patients with chronically high immune activation.

Comparative Approach to Define Increased Regulatory T Cells in Different Cancer Subtypes by Combined Assessment of CD127 and FOXP3

In recent years an increase of functional CD4+CD25+ regulatory T cells (Treg cells) has been established for patients with solid tumors, acute leukemias, and lymphomas. We have reported an expanded pool of CD4+CD25high Treg cells in patients with chronic lymphatic leukemia (CLL), multiple myeloma (MM) as well as its premalignant precursor monoclonal gammopathy of undetermined significance (MGUS). In healthy individuals, low-level expression of CD127 on T cells in addition to the expression of FOXP3 has been associated with Treg cells. Here, we demonstrate that the expanded FOXP3+ T-cell population in patients with colorectal cancer, CLL, MGUS, MM, follicular lymphoma, and Hodgkin's disease are exclusively CD127low Treg cells and were strongly suppressive. A significant portion of CD127lowFOXP3+ Treg cells expressed only low levels of CD25 suggesting that the previously reported expansion of CD25+ Treg cells underestimates the true expansion. The assessment of CCR7 and CD45RA expression on the expanded CD4+CD127lowFOXP3+ Treg cells revealed an increase of both naïve as well as central and effector memory Treg cells in peripheral blood. Our data strongly support superiority of combined CD127 and FOXP3 analysis in comparison to CD25 and FOXP3 assessment for further quantification of Treg cells in malignant diseases.

Emergence of Dominant T-Cell Populations in Patients with CLL after Allogeneic Transplantation: Mediators for Gvl Effects?

Allogeneic stem cell transplantation (SCT) is the only known curative treatment for high-risk CLL. We have recently shown that minimal residual disease (MRD) monitoring can identify patients with graft versus leukemia (GvL)-induced disease response to either reduction of immunosuppression (IS) or to administration of donor lymphocyte infusions (DLI), suggesting that those patients are potentially cured by an ongoing immunologic antileukemic effect induced by donor immune cells (Leukemia 22:1377). It is uncertain, however, which cell population maintains this process; although T as well as NK-cell mediated effects are discussed. The present study addressed the question whether disease response upon immunomodulation after SCT is associated with the occurrence of dominant T cell clones.Methods: 32 patients allografted for high-risk CLL who had MRD follow-up by clone-specific PCR or MRD-flow available were included in this investigation. We used the BIOMED T-cell receptor multiplex PCRs (TCR-PCR) to search for T cell clones which might be involved in the documented GVL effects. TCR rearrangements were sequenced and analyzed using the IMGT database.Results: 16 of 32 patients showed MRD response after IS reduction or DLI. GVL-induced MRD clearance was associated with onset of chronic GVHD in almost all instances. Twenty-four different dominant TCR rearrangements could be identified in 15/32 patients by BIMOD TCR-PCR. Most of the T cell populations show rearranged gamma/delta TCRs suggesting that regulatory gamma/delta T cells might be involved in this process. TCR sequences employed were TRGV9 (13), TRGV2 (2) and TRGV1, TRGV4, TRGV8, TRGV10, TRGV11, TRBV5, TRBV6, TRBV12, TRBV15. In 4 patients with a potential productive TCR rearrangement (TRGV4+TRDV1, TRBV6, TRGV2, TRGV11+TRGV9) we were able to design a TCR-specific real-time PCR for quantitative follow-up of this clonal T cell population. This data was compared to flow cytometric monitoring of T-cell subpopulations and MRD kinetics post SCT. In those 4 patients we could demonstrate an inverse correlation of the kinetics of MRD and the kinetics of clonal T cell expansions. T cell clones emerging during this phase remained on a stable level throughout the whole follow-up in patients showing durable MRD negativity.Conclusion: In CLL, MRD clearance after SCT is correlated to the emergence of dominant T cell clones, suggesting that GVL activity is based on allo- or CLL-specific T cell expansion. Further studies are needed to clarify the role of these T cell clones for GVL and GVHD development.

Interferon-α: A Key Factor in Autoimmune Disease?

Interferon (IFN)-alpha is an effective drug for treatment of uveitis in Behçet's disease. This study was undertaken to investigate the mechanism of action of IFN-alpha in the treatment of various types of noninfectious sight-threatening uveitis. Eleven patients with refractory uveitis, and 13 healthy individuals were enrolled. The number of circulating plasmacytoid dendritic cells (pDCs) and their capacity to produce IFN-alpha in culture on stimulation with synthetic oligodinucleotides containing the CpG-motif were studied. Peripheral blood CD4+ T-cell phenotype and activation status were evaluated by flow cytometry at 0, 2, and 8 weeks after treatment for expression of CD69, CD62L, chemokine receptors (CCR4, CXCR3, and CCR5), and intracellular cytokines (TNF-alpha, IFN-gamma, and IL-10). All patients experienced a positive clinical response to IFN-alpha treatment. There was no significant difference between patients and control subjects in the number of circulating pDCs, but there was a significant decrease in the capability of patients' pDCs to produce IFN-alpha in response to CpG (P < 0.001). Peripheral blood CD4+ T cells expressed reduced levels of surface CD62L (P < 0.005) as a measure of activation and higher levels of chemokine receptors CXCR3, CCR4, and CCR5 (P < 0.005, P < 0.05, and P < 0.05, respectively); in addition, intracellular T-cell IL-10 levels were increased once the treatment was initiated (P < 0.01). The data suggest that IFN-alpha may control uveitis by promoting induction of IL-10-producing T-cells, possibly T-regulatory cells. Dysregulation of the T-cell population in patients with uveitis may be associated with a defect in the pDCs' ability to produce IFN-alpha, which can be circumvented with administration of exogenous IFN-alpha.

Alterations in the T-Cell Receptor Variable β Gene–Restricted Profile of CD8+ T Lymphocytes in the Peripheral Circulation of Patients with Squamous Cell Carcinoma of the Head and Neck

Apoptosis of activated CD8(+) T cells is often seen in tumor-infiltrating lymphocytes and circulating peripheral blood mononuclear cells (PBMC) in patients with squamous cell carcinoma of the head and neck (SCCHN). We investigated whether T-cell receptor (TCR) variable beta chain (Vbeta)-restricted T cells were more sensitive to apoptosis than non-TCR Vbeta-restricted T cells. Flow cytometry analysis with anti-TCR Vbeta antibodies was used to define expansions and contractions of Vbeta-restricted T cells in patients with SCCHN relative to normal donors. This staining was combined with Annexin V binding to indicate early T-cell apoptosis. The TCR Vbeta profiles of CD3(+) T cells in tumor-infiltrating lymphocytes and PBMCs of patients with SCCHN were altered relative to controls, with one to five expansions and numerous contractions of TCR Vbeta-restricted T cells detected. These types of alterations were significantly greater in CD8(+) than CD4(+) T cells. Enhanced Annexin V binding to CD8(+) T cells was evident in PBMCs obtained from all patients, with 3 of 13 showing preferential targeting for apoptosis of TCR Vbeta-restricted T cells. TCR Vbeta profiles of CD8(+) T cells were altered in patients with SCCHN relative to normal controls. This may reflect increased apoptosis of expanded or contracted CD8(+) T cells, which define the TCR Vbeta profile of antigen-responsive T-cell populations in patients with cancer.

T-cell homeostasis in humans with thymic hypoplasia due to chromosome 22q11.2 deletion syndrome

AbstractPatients with chromosome 22q11.2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome) typically exhibit thymic hypoplasia, conotruncal cardiac defects, and hypoparathyroidism. The immunodeficiency that results from the thymic hypoplasia has been extensively described and consists primarily of T-cell lymphopenia. A curious feature of the T-cell lymphopenia is that the age-related rate of decline of T-cell numbers is slower in patients than controls. This leads to T-cell numbers in adulthood that are minimally decreased compared with controls. This suggests that homeostatic mechanisms might be acting to preserve the peripheral blood T-cell numbers in patients. We characterized changes in CD4/CD45RA and CD4/CD45RO T-cell populations in patients and controls of various ages and determined T-cell recombination excision circles and telomere length within the CD4/CD45RA population. Patients had evidence of accelerated conversion of naive to memory cells and had evidence of more extensive replicative history within the CD4/CD45RA compartment compared with controls. Oligoclonal T-cell receptor (TCR) Vβ families and missing Vβ families were seen more often in patients than controls. These data are consistent with homeostatic proliferation of T cells in patients with limited T-cell production due to thymic hypoplasia. (Blood. 2004;103:1020-1025)

Identification of clonal T cells in the blood of patients with systemic sclerosis: positive correlation with response to photopheresis.

To search for circulating clonal T-cell populations in patients with systemic sclerosis (SSc), and to determine whether T-cell clonality in the blood predicts therapeutic response to photopheresis. Analysis of clonal T-cell receptor gamma gene rearrangements before photopheresis treatment and blinded clinical evaluation of cutaneous response to photopheresis in a case series. University hospital setting. Thirteen consecutive patients with SSc. Photopheresis in 11 patients. Clonality of T cells in the blood before photopheresis and clinical response to photopheresis. Screening of blood samples from 13 SSc patients for clonal T-cell receptor gamma gene rearrangements revealed a monoclonal T cell population in 6 (46%) of 13 SSc patients. Clinical response to photopheresis in 11 patients was evaluated in a blinded manner using skin severity scores. Clonality of T cells appeared to be associated with a higher chance of response to photopheresis therapy, as 4 (67%) of 6 patients in the clone-positive group vs 1 (20%) of 5 in the clone-negative group experienced a clinically significant response to treatment. A high proportion of patients with SSc have detectable expanded clonal T-cell populations in the peripheral blood, and such patients appear more likely to respond to photopheresis.

Disease-dependent changes in T-cell populations in patients with shigellosis.

In shigellosis, bacterial infection is associated with an extensive inflammation of the rectal mucosa, resulting in bloody dysentery. The role of T-cell-mediated pro-inflammatory mechanisms has been implicated in this process, but the specific role of T-cell subsets is still not well understood. In this study we attempted to identify the changes in T-cell populations in patients with shigellosis during the disease course. The T-cell subset distribution was analyzed by immunohistochemistry in the rectal mucosa and by immuno-flow cytometry in the peripheral blood. Blood and rectal biopsies were studied from patients with Shigella dysenteriae 1 (n= 11) and S. flexneri (n= 11) infection and 20 healthy age-matched controls. We found an expansion of gammadelta+T cells in the rectal mucosa, but a decrease in the percentage of gammadelta+T cells in the blood in acute shigellosis. There was also a preferential increase in CD8+ T cells in the surface epithelium of rectal tissue in patients infected with S. dysenteriae 1, but not in patients infected with S. flexneri. Our findings suggest that the rectal mucosal inflammation in shigellosis is associated with an expansion of T cells, in particular CD8+ and gammadelta+T-cell subsets in the gut mucosa, which may be of importance for the pathogenesis of shigellosis.

Cellular reaction to group A beta-haemolytic streptococcal membrane antigen and its relation to complement levels in patients with rheumatic heart disease.

Cell-mediated immunity and blood complement activities were studied in 35 patients with chronic rheumatic heart disease (RHD) and 17 normal subjects. The T-cell population in patients with RHD was reduced, as were the CH50 and C3 complement levels. The response to phytohaemagglutinin stimulation was deficient, but the lymphocytes of patients with RHD showed increased avidity for 3H-thymidine when stimulated with specific streptococcal membrane antigen. No differences were found between patients with acute rheumatic activity and those without such activity. The susceptibility of individual patients may be related to the specific sensitisation of lymphocytes, while the fact that this persisted even when T-cell numbers had returned to normal may account for the well-known recrudescenses after streptococcal infections in these patients.

ISOLATION OF NORMAL T CELLS IN CHRONIC LYMPHATIC LEUKÆMIA

Lymphocytes from patients with chronic lymphatic leukæmia (C.L.L.) show a poor response to mitogens. Lymphocytes from these patients and from healthy patients were separated into two populations. One population, very rich in T cells, showed the same type of response for both the controls and patients. In contrast, the population poor in T cells derived from C.L.L. lymphocytes did not respond at all to mitogens. Recombination experiments indicate that the response can be restored by the addition of T cells. These findings demonstrate the presence of a normally reactive T-cell population in patients with C.L.L. It seems that the lack of responsiveness in C.L.L. is due to the dilution of normally responding cells.