Low Numbers Of CD4 Research Articles

Regulatory T cells, interleukin (IL)‐6, IL‐8, Vascular endothelial growth factor (VEGF), CXCL10, CXCL11, epidermal growth factor (EGF) and hepatocyte growth factor (HGF) as surrogate markers of host immunity in patients with renal cell carcinoma

To identify a phenotype that could be informative and prognostic in patients with renal cell carcinoma (RCC) peripheral blood was evaluated for TH1, TH2, regulatory T cells (Tregs), natural killer (NK) and NKT cells and for cytokines/chemokines. Peripheral blood from 77 patients with RCC and 40 healthy controls was evaluated by flow cytometry using monoclonal antibodies against CD4, CD25, FoxP3, CD45RA, CD45RO, CD152, CD184, CD279, CD3, CD16, CD56, CD161, CD158a, CD4, CD26, CD30, CD183 and CD184. A concomitant evaluation of 38 molecules was conducted in patients' serum using a multiplex biometric ELISA-based immunoassay. The number of NK cells CD3⁻/CD16⁺, CD3⁻/CD16⁺/CD161⁺ (NK) and CD3⁻/CD16⁺/CD161⁺/CD158a⁺ (NK- Kir 2+) was greater in the patients with RCC (P < 0.05); and the number of Treg cells CD4⁺/CD25(high+)/FOXP3⁺ and the subset CD4⁺/CD25(high+)/FOXP3⁺/CD45RA⁺ (naïve) and CD45R0⁺(memory) cells, were greater in the patients with RCC (P < 0.001). An increase in the following was observed in the serum of patients with RCC compared with healthy controls: interleukin (IL)-4, IL-6, IL-8, IL-10, G-CSF, CXCL10, CXCL11, hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF). According to Ingenuity Pathway Analysis (IPA), CXCL10, IL-6, IL-8, epidermal growth factor (EGF), HGF and VEGF were associated with a network that controls cellular movement, tissue development and cellular growth. Kaplan-Meier analysis for disease-free survival showed that high numbers of CD4⁺/CD25(high+)/FOXP3⁺/CD45RA⁺ (Treg naïve) and low numbers of CD3⁻/CD16⁺/CD161⁺/CD158a⁺ (NK-Kir+) cells predict short disease-free survival in patients with RCC. Concomitant evaluation of Treg (CD4⁺/CD25(high+)/FOXP3⁺ and CD4⁺/CD25(high+)/FOXP3⁺/CD45RA⁺) and of six soluble factors (IL-6, IL-8 ,VEGF, CXCL10, CXCL11, EGF, HGF) might be a surrogate marker of host immunity in patients with RCC.

Diagnosis and evaluation of intestinal graft-versus-host disease after allogeneic hematopoietic stem cell transplantation following reduced-intensity and myeloablative conditioning regimens

Colonoscopic evaluation of mucosal tissues after allogeneic hematopoietic stem cell transplantation (HSCT) is very useful in evaluating pathogenesis and diagnosis of intestinal graft-versus-host disease (GVHD). However, information on the timing and sites of biopsies and the immunohistological evaluation of mucosal tissues for diagnosing intestinal GVHD, especially following reduced-intensity (RIC) regimens, remains very limited. A total of 33 patients with histologically proven GVHD after allogeneic HSCT with RIC (n = 23) and myeloablative conditioning (MAC, n = 10) regimens were enrolled in the present study. Colonoscopy was performed due to gastrointestinal symptoms, especially diarrhea and anorexia. Sites of biopsies with the worst histopathological grading were the terminal ileum in 67 % of patients. In the RIC group, the onset of diarrhea prior to colonoscopy examination was later (median: RIC, 57 vs. MAC, 27 days) and the number of patients who developed abdominal pain tended to be higher (RIC, 70 % vs. MAC, 30 %). A lower number of CD4+ cells and a higher ratio of Foxp3+ cells to CD4+ cells were detected in the involved lesions of intestinal GVHD following RIC. These differences in the RIC and MAC groups suggest that regimen-specific therapeutic strategies are required for diagnosing intestinal GVHD.

The differences in T and B cell subsets in thyroid of children with Graves’ disease and Hashimoto’s thyroiditis

The differences between Graves' disease (GD) and Hashimoto's thyroiditis (HT) suggest that changes in the subsets of T cells may have an influence on the course of these reactions. This study included 90 children: 30 with GD, 30 with HT, and 30 healthy children as controls. After thyroidectomy, standard histological examinations and immunohistochemical reactions were performed in paraffin specimens with monoclonal antibodies against T cell markers CD3, CD4, CD8 as well as against CD79 alpha B cells. Ultrathin sections were examined under a transmission electron microscope. Autoimmune reaction in GD consisted of an increased number of CD4+ T cells (3.17±4.27%) and plasma cells (22.89±8.61%) producing thyroidstimulating hormone-receptors and stimulating thyrocytes to activity. The number of CD8+ T cells was increased in children with HT (20.54±0.68%) as compared with the controls (0.65±0.30%). The autoimmune reaction in the HT children showed antibody dependent cytotoxicity with a low number of CD4+ T cells and an increased number of CD8+ T cells in the thyroid tissue in comparison with that in the GD children and the controls. Plasma cells (31.65±9.11%) in this situation produced the antibodies involved in cytotoxic reactions against thyrocytes. Graves' disease is characterized by the increased number of CD4+ T cells and CD8+ T cells. Hashimoto's thyroiditis is characterized by the low number of CD4+ T cells and increased number of CD8+ T cells. CD8+ T cells have cytotoxic properties only in Hashimoto's thyroiditis.

Abstract B23: DNA damage response in responder lymphocytes reduces vaccine efficacy following alkylating chemotherapy.

Abstract The effect of alkylating chemotherapy on cancer vaccines is controversial, with numerous reports of immune stimulation via Treg depletion following cyclophosphamide and temozolomide treatment. Using a pre-clinical model of a cancer vaccine in both naïve and tumor bearing animals, we observed that the direct anti-proliferative effect of drugs such as temozolomide or cyclophosphamide reduced the quantity of vaccine induced immune responses in a dose dependent manner. Amounts of temozolomide exposure in mice that duplicated human pharmacokinetic exposure and lymphopenia led to lower numbers of CD8 T cells elicited by vaccination with the model antigen ovalbumin. In vitro experiments with purified OT-I CD8 T cells implicated activation of DNA damage responses within strongly proliferating cells as the cause of this diminished immunity. ATM phosphorylation was greater in temozolomide or cyclophosphamide exposed OT-I cells stimulated with the canonical SIINFEKL peptide relative to variant peptides that trigger weaker TCR signaling. In vaccinated mice, this activation of DNA damage response in lymphocytes receiving the highest intensity TCR signals lead to lower avidity of responder cells for cognate antigen and lower effector function. These quantitative and qualitative defects of vaccine driven responses lead to inferior efficacy and loss of survival benefit in murine models. Mice bearing ovalbumin peptide expressing glioma had faster tumor growth and shorter survival following temozolomide and vaccine relative to vaccine only treated mice. Similar defects of vaccine generated responses and a loss of survival benefit relative to controls were observed in response to a neo-antigen vaccine following cyclophosphamide pre-treatment in melanoma bearing mice. The clinical relevance of this finding is demonstrated by a phase II cancer vaccine trial in metastatic melanoma patients where vaccine only patients survived a median of 4.2 years versus less than 8 months in patients treated with low dose cyclophosphamide before vaccine. Citation Format: Adam J. Litterman, David M. Zellmer, Karen L. Grinnen, Arkadiusz Z. Dudek, David A. Largaespada, John R. Ohlfest. DNA damage response in responder lymphocytes reduces vaccine efficacy following alkylating chemotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B23.

Elevated Gamma Delta T Cell Recovery Following Hematopoietic Stem Cell Transplantation Associated with Improved Long Term Overall Survival in Pediatric Patients with Acute Leukemia

Abstract 227Recent studies show that accelerated γΔ T cell reconstitution after hematopoietic stem cell transplantation (HSCT) is associated with improved overall survival (OS) though the mechanisms have not been elucidated. Here, we confirm that early γΔ T cell recovery after HSCT is an independent predictor for improving OS and event free survival (EFS). Moreover, patients with robust γΔ T cell recovery after HSCT appear to be protected from increased risk of serious life threatening infections, and acute gut and chronic graft versus host disease (GVHD). More importantly, we show that γΔ T cell recovery dynamics are independent from those of classical αβ T cells. We evaluated 102 consecutive pediatric patients with acute leukemia undergoing HSCT at St. Jude Children’s Research Hospital from 1996–2011. The median age of the patients was 10.5 ± 5.9 yrs. (range, 0.6–25.2) and median follow up was 2.7±1.8 yrs. (range 0.2–6.0). There were 57% males, 43% females and 59% with ALL and 41% with AML. There were 14 patients with elevated γΔ T cells (≥1.75×105 cells/ml) and 88 with low/normal γΔ T cells (<1.75×105 cells/ml). There were no significant differences between the two groups with respect to age, sex, disease or donor source, p=0.7, 0.5, 1 and 0.07 respectively. Fours years after HSCT, OS was significantly higher for patients in the elevated group compared to the patients in the low/normal group, 93% and 60%, respectively, p=0.0173. Survival without relapse or graft failure (EFS) was significantly higher in the elevated group compared to the low/normal group, 85.7% and 58.0%, respectively. Since T cell reconstitution following HSCT is age dependent, we determined if γΔ T cell recovery correlated with age and/or CD3+ cells. Multivariate analysis showed no correlation between the number of CD3+ and γΔ T cells. In fact, 13 of 14 patients that recovered with increased number of γΔ T cells had normal or low numbers of CD3+ cells. Thus, γΔ T cell recovery is not a simple correlate of T cell reconstitution. Because γΔ T cells play a central role in maintaining intestinal epithelium integrity, we evaluated the incidence of gut GVHD. We found a significant lower rate of gut GVHD in the elevated group compared to the low/normal group, 0% and 17% respectively. Furthermore, the number of γΔ T cells in patients with cGVHD (2.3 x105 cells/ml) was significantly lower compared to patients without cGVHD (6.2 x105 cells/ml), p=0.01. This suggests that γΔ T cell may protect against gut and cGVHD. Since accumulating evidence suggests that γΔ T cells contribute to both innate and adaptive immune responses during infections, we evaluated the rate and types of infection between the two groups. We found a significant lower incidence of infection in the elevated group compared to the low/normal group, 21% and 54% respectively p=0.02. Furthermore, the elevated group had only viral infections while the low/normal group had viral, bacterial and fungal infections. Recent studies suggested that γΔ T cells could contribute to surveillance of CMV reactivation after HSCT through cooperation with anti-CMV IgG. Evaluation of CMV infections found no significant decrease in the incidence of CMV infection in the elevated group compared to the low/normal group, 14% and 2%. In summary, this is the first reported study of γΔ T cell recovery after HSCT in pediatric patients and adds new insights into the role γΔ T cells by evaluating the relationship of the most common complications such as relapse, GVHD and infections. Disclosures:No relevant conflicts of interest to declare.

Epstein-Barr virus Peptide Presented by HLA-E is Predominantly Recognized by CD8bright Cells in multiple Sclerosis Patients

Multiple sclerosis (MS) is associated with Epstein-Barr virus (EBV) infection, but impaired immune suppression may be part of the disease pathogenesis. CD8+ T cells that are restricted by HLA-E exert an important immunoregulatory mechanism. To explore how EBV might interfere with immune regulation, we examined the expression of HLA-E and the frequency of CD8+ cells recognizing HLA-E, presenting either an EBV peptide from the BZLF1 protein or a signal sequence peptide from HLA-A2, in relapsing remitting (MS-RR), primary progressive (MS-PP) MS patients, and healthy controls (HC). Treatment with IFN-α or EBV increased HLA-E expression on CD4+ cells. However, only MS-PP had increased expression of HLA-E on resting CD4+ cells when compared with HC (p<0.005). CD8+ cells were divided into CD8bright and CD8dim cells by flow cytometry analyses. MS-RR had significantly fewer CD8dim cells than HC (p<0.003). Flow cytometry analyses were performed with HLA-E tetramers folded in the presence of the EBV or HLA-A2 peptide to identify HLA-E-interacting cells. MS-RR had increased frequency of CD8bright cells recognizing HLA-E/A2 (p = 0.006) and HLA-E/BZLF1 (p = 0.016). Conversely, MS-RR had fewer CD8dim cells that recognized HLA-E/BZLF1 (p = 0.001), but this could be attributed to the overall lower number of CD8dim cells in MS-RR. Whereas HLA-E/A2 was predominantly recognized by CD8dim cells, HLA-E/BZLF1 was predominantly recognized by CD8bright cells in MS-RR and MS-PP, but not in HC. As expected, HLA-E/A2 was also recognized by CD8-negative cells in a CD94-dependent manner, whereas HLA-E/BZLF1 was poorly recognized in all groups by CD8-negative cells. These data demonstrate that MS-RR patients have expanded their CD8bright cells recognizing HLA-E/BZLF1. Moreover, HLA-E/BZLF1 appears to be recognized by the immune system in a different manner than HLA-E/A2.

An MHC Class Ib-Restricted CD8+ T Cell Response to Lymphocytic Choriomeningitis Virus

Conventional MHC class Ia-restricted CD8(+) T cells play a dominant role in the host response to virus infections, but recent studies indicate that T cells with specificity for nonclassical MHC class Ib molecules may also participate in host defense. To investigate the potential role of class Ib molecules in anti-viral immune responses, K(b-/-)D(b-/-)CIITA(-/-) mice lacking expression of MHC class Ia and class II molecules were infected with lymphocytic choriomeningitis virus (LCMV). These animals have a large class Ib-selected CD8(+) T cell population and they were observed to mediate partial (but incomplete) virus clearance during acute LCMV infection as compared with K(b-/-)D(b-/-)β(2)-microglobulin(-/-) mice that lack expression of both MHC class Ia and class Ib molecules. Infection was associated with expansion of splenic CD8(+) T cells and induction of granzyme B and IFN-γ effector molecules in CD8(+) T cells. Partial virus clearance was dependent on CD8(+) cells. In vitro T cell restimulation assays demonstrated induction of a population of β(2)-microglobulin-dependent, MHC class Ib-restricted CD8(+) T cells with specificity for viral Ags and yet to be defined nonclassical MHC molecules. MHC class Ib-restricted CD8(+) T cell responses were also observed after infection of K(b-/-)D(b-/-)mice despite the low number of CD8(+) T cells in these animals. Long-term infection studies demonstrated chronic infection and gradual depletion of CD8(+) T cells in K(b-/-)D(b-/-)CIITA(-/-) mice, demonstrating that class Ia molecules are required for viral clearance. These findings demonstrate that class Ib-restricted CD8(+) T cells have the potential to participate in the host immune response to LCMV.

Involvement of Indoleamine 2,3-Dioxygenase in Impairing Tumor-Infiltrating CD8+T-Cell Functions in Esophageal Squamous Cell Carcinoma

The indoleamine 2,3-dioxygenase-(IDO-) mediated microenvironment plays an important role in tumor immune escape. However, the inhibitory effects of IDO on the CD8+ tumour-infiltrating lymphocytes (CD8+ TILs) in esophageal squamous cell carcinoma (ESCC) have not been clarified yet. Here, we found that the level of IDO expression in ESCC tumor specimens correlated with a reduction in the number of CD8+ TILs. Patients with high IDO expression and a low number of CD8+ TILs had significantly impaired overall survival time. IDO expression and functional enzyme activity in ESCC cell lines could be induced by IFNγ. When exposed to the milieu generated by IDO-expressing Eca109 cells, the CD8+ TILs were suppressed in proliferation, and their cytolytic functions against target tumor cells were lost. These results suggested that impairing CD8+ TIL functions by IDO expressed in ESCC possibly contributed to the finding that patients with higher IDO expression have more aggressive disease progression and shorter overall survival time.

HSCT PATIENTS with HHV6 Reactivation HAD LOW NUMBERS of CD4+ Effector Memory Lymphocytes IN BLOOD WHAT Associates with Rather POOR SURVIVAL.

AbstractAbstract 4555It is known that the pace of immunological recovery influences the outcome of HSCT and herpes viruses reactivation contributes significantly to the post transplant mortality.The aim of this study was to investigate reconstitution of subpopulations of CD4+ lymphocytes in the context of β-herpes viruses reactivation shortly post HSCT. There were two observation points: (1) at the beginning of lymphocyte recovery (number of lymphocytes > 200 cells/ul) which was seen from 9 to 31 day post HSCT median 14 and (2) 3 weeks later (median day 35 range(31-53)). CD4+ T cell subsets were defined as follow: Naive cells (N) CD4+CCR7+CD45RA+, Central memory (CM) CD4+CCR7+CD45-, Effector memory(EM) CD4+CCR7-CD45+, Terminally differentiated memory cells (TEMRA) CD4+CCR7-CD45RA+ . CMV, EBV and HHV-6 DNA copies were detected in blood with the use of QT-PCR in one week intervals. The study included 44 patients (age 1 to 64 median 45yr., 21 ric/mac, 21/23, sib/mud 21/23 mud, BM/PBPC 4/40.It was found;1. Fifteen, 13 and 4 patients were positive for EBV, HHV-6 and CMV DNA copies during the observation period.(i) CD4+lymphocyte count was lower in patients having CMV copies as compared to those having EBV and/or HHV6 copies (both lymphocytotropic viruses) at the beginning of lymphocytes recovery (45/ul vs 120/ul p=0,047 Mann-Whitney U test), similarly behaved EM cells. 22/ul vs 84/ul EM cells (p= 0,088 Mann-Whitney U test) were seen in pts having CMV reactivation and in those with EBV and/or HHV-6 reactivation, respectively.(ii) The situation changed three weeks later, it appeared that patients having HHV6 reactivation any time during the observation period had lower CD4+ lymphocyte numbers as compared to patients lacking β-herpes viruses reactivations (CD4+ cells median 93/ul vs190/ul; p=0,096 Mann-Whitney U test) and to those with CMV and EBV reactivation. Again EM cells contributed greatly to these differences. EM CD4+ lymphocytes were 41/ul, 105/ul, 80/ul and 160/ul in patients having HHV-6, CMV or EBV and lacking beta-herpes viruses reactivation, respectively. EM CD4+ lymphocyte numbers for HHV6+ patients as compared to patients lacking β-herpes viruses reactivations was 41/ul vs155/ul.(p=0,012 Mann-Whitney U test).Conclusions: It appears that HHV6 is the main player in shaping lymphocyte reconstitution post transplant affecting predominantly EM cells what influences the outcome of transplantation. In this group of patients probability of one year survival was 50% vs 65%, in patients having and lacking HHV6, respectively. Disclosures:No relevant conflicts of interest to declare.

Determination of CD4+ and CD8+ T cells in the peripheral blood of dogs with demodicosis

The aim of this study was to evaluate the CD4+/CD8+ ratio in peripheral blood of dogs with localized and generalized demodicosis. Sixteen dogs were examined, 8 with localized and 8 with generalized demodicosis, while 8 healthy dogs were used as controls. Peripheral blood was obtained and CD4+ and CD8+ T cells were determined by flow cytometry. Significantly higher numbers of CD8+ T cells and lower numbers of CD4+ T cells were found in dogs with generalized demodicosis compared to dogs with localized demodicosis and healthy controls. Significantly higher numbers of CD8+ T cells and lower numbers of CD4+ T cells were also found in dogs with localized demodicosis compared to healthy controls. The CD4+/CD8+ ratio was also found to be significantly lower in dogs with generalized demodicosis in comparison with dogs with localized demodicosis and healthy controls. It is concluded that significant alteration in the CD4+/CD8+ ratio may be implicated in the pathogenesis of generalized canine demodicosis.

Post-transplant immune reconstitution after unrelated allogeneic stem cell transplant in patients with acute myeloid leukemia

We evaluated immune recovery in 67 patients with acute myeloid leukemia (AML) with a median age of 40 years (4–69) following allo-SCT after reduced (n = 35) or myeloablative (n = 32) conditioning. The following lymphocyte populations were determined on days +30, +90, +180, +270, and +365 by flow associated cell sorting: CD3+, CD3+CD4+, CD3+CD8+, CD3+CD4+/CD3+CD8+ ratio, CD3−CD56+, and CD19+ cells. Peripheral blast count >5% was related to lower number of CD3+CD4+ (day +30) and NK cells (day +180; p = 0.02). Intensity of conditioning did not have any significant impact on the kinetics of immune recovery. Patients with normal CD3+CD4+/CD3+CD8+ ratio (day +30) and NK cell count (day +90; p <0.05) experienced better survival than those with decreased parameters. Post-transplant sepsis/severe infections impaired CD3+CD8+ (day +90; p = 0.015) and CD19+ (day +90; p = 0.02) recovery. Relapse in patients following allo-SCT showed an association with decreased numbers of CD19+ (day +270) and NK cells (day +365). Acute GvHD (II–IV) was accompanied by reduced CD19+ and CD3+CD4+ cells. Thus, the evaluation of post-transplant immune reconstitution in patients with AML might improve risk stratification concerning either relapse or TRM and remains to be further explored.

Leptin Modulates Innate and Adaptive Immune Cell Recruitment after Cigarette Smoke Exposure in Mice

Leptin, a pleiotropic type I cytokine, was recently demonstrated to be expressed by resident lung cells in chronic obstructive pulmonary disease patients and asymptomatic smokers. To elucidate the functional role of leptin in the onset of chronic obstructive pulmonary disease, we tested leptin-deficient ob/ob mice (C57BL/6), leptin receptor-deficient db/db mice (C57BKS), and littermates in a model of cigarette smoke (CS)-induced pulmonary inflammation. Wild-type (WT) C57BL/6 mice were exposed for 4 or 24 wk to control air or CS. Pulmonary leptin expression was analyzed by immunohistochemistry and real-time PCR. Pulmonary inflammation upon 4 wk CS exposure was evaluated in bronchoalveolar lavage fluid (BALF) and lung tissue of WT, ob/ob, and db/db mice. Immunohistochemical analysis revealed leptin expression in bronchial epithelial cells, pneumocytes, alveolar macrophages, and bronchial/vascular smooth muscle cells. The 4 and 24 wk CS exposure increased leptin expression in bronchial epithelial cells and pneumocytes versus air-exposed WT mice (p<0.05). The 4 wk CS exposure resulted in increased accumulation of neutrophils, dendritic cells, macrophages, and lymphocytes in BALF and lung tissue of WT, ob/ob, and db/db mice. CS-exposed ob/ob and db/db mice showed in general higher numbers of neutrophils and lower numbers of CD4+, CD8+, and dendritic cells versus CS-exposed WT mice. Consistently, CXCL1 levels were enhanced in BALF of CS-exposed ob/ob and db/db mice versus WT mice (p<0.05). Exogenous leptin administration completely restored the skewed inflammatory profile in ob/ob mice. These data reveal an important role of leptin in modulating innate and adaptive immunity after CS inhalation in mice.

Differential monocyte STAT6 activation and CD4 +CD25 +Foxp3 + T cells in kidney operational tolerance transplanted individuals

In organ transplantation, the immunosuppression withdrawal leads, in most cases, to rejection. Nonetheless, a special group of patients maintain stable graft function after complete withdrawal of immunosuppression, achieving a state called “operational tolerance.” The study of such patients may be important to understand the mechanisms involved in human transplantation tolerance. We compared the profile of CD4 +CD25 +Foxp3 + T cells and the signaling pathways IL-6/STAT3 (signal transducers and activators of transcription) and IL-4/STAT6 in peripheral blood mononuclear cells of four kidney transplant groups: (i) operational tolerance (OT), (ii) chronic allograft nephropathy (CR), (iii) stable graft function under standard immunosuppression (Sta), (iv) stable graft function under low immunosuppression, and (v) healthy individuals. Both CR and Sta displayed lower numbers and percentages of CD4 +CD25 +Foxp3 + T cells compared with all other groups ( p < 0.05). The OT patients displayed a reduced activation of the IL-4/STAT6 pathway in monocytes, compared with all other groups ( p < 0.05). The lower numbers of CD4 +CD25 +Foxp3 + T cells observed in CR individuals may be a feature of chronic allograft nephropathy. The differential OT signaling profile, with reduced phosphorylation of STAT6, in monocytes' region, suggests that some altered function of STAT6 signaling may be important for the operational tolerance state.

Low numbers of CD8+ T lymphocytes in hereditary haemochromatosis are explained by a decrease of the most mature CD8+ effector memory T cells

Low CD8(+) T lymphocyte numbers have long been described in hereditary haemochromatosis (HH). Recently, two conserved haplotypes localized near the microsatellite D6S105 at the major histocompatibility complex (MHC) class I region were described predicting the clinical expression of HH and the CD8(+) T lymphocyte numbers. The A-A-T haplotype was associated with a severe clinical expression of HH and low CD8(+) T lymphocyte numbers, while the G-G-G haplotype was associated with a milder clinical expression of HH and high CD8(+) T lymphocyte numbers. As CD8(+) T lymphocytes are a very heterogeneous population, in this study we analysed the CD8(+) subpopulations of naive, central memory (T(CM)) and effector memory (T(EM)), and further subsets of CD8(+) T(EM) cells in 47 HH patients and 68 controls. In addition, association studies were conducted between the conserved haplotypes and the CD8(+) T cell subpopulations in HH. Variations of the numbers of naive and central memory cells with age were similar between HH patients and controls. For T(EM) cells and the T(EM) CD27(-)CD28(-) subset no effect of age was observed in HH [R(2) = 0.001, not significant (n.s.) and R(2) = 0.01, n.s., respectively] contrasting with the increasing of these subpopulations with age in controls (R(2) = 0.09, P = 0.017 and R(2) = 0.22, P = 0.0005, respectively). Interestingly, patients homozygous for the A-A-T haplotype have lower numbers of CD8(+) T(EM) cells due especially to lower numbers of T(EM) CD27(-)CD28(-) (0.206 +/- 0.119 and 0.066 +/- 0.067 x 10(6) cells/ml, respectively) than patients carrying the G-G-G haplotype (0.358 +/- 0.195 and 0.246 +/- 0.202 x 10(6) cells/ml, respectively). This may suggest an inability of HH patients to differentiate the CD8(+) T cells into the most mature phenotype.

Memory CD8. + T Cells Specific for Minor Histocompatibility Antigen H60 Can Mediate the Graft-Versus-Leukemia Effect.

Abstract 1332Poster Board I-354In allogeneic hematopoietic stem cell transplantation (alloSCT), donor T cells mediate graft-vs-leukemia (GVL) but also cause graft-vs.-host disease (GVHD). Our previous studies indicate that memory T cells (TM) induce less GVHD than do naïve T cells (TN). Therefore the selective infusion of memory T cells could improve immune reconstitution with less GVHD. However, human memory T cells have a more restricted TCR repertoire than do TN and therefore may be less effective in mediating GVL. One approach for addressing this concern would be to vaccinate donors against a single miHA expressed by host leukemic cells. Then the selective transfer of memory T cells could improve both GVL and immune reconstitution with less GVHD. Questions remain about how best to apply this strategy. For example, differences in target antigen distribution could affect the re-expansion of transferred memory cells and determine whether vaccination augments GVHD. Also, different types of memory cells could behave differently. To begin to optimize this approach in mouse models we chose as our target antigen the H-2Kb-restricted minor histocompatibility antigen (miHA) H60. We cloned the H60 peptide sequence (LTFNYRNL) into a genetic construct encoding the heavy chain of a monoclonal antibody against DEC205. Donor C3H.SW (H-2b, H60-) mice were vaccinated with a single injection of anti-DEC205-H60 plus an agonist antibody against CD40 (FGK45). By two months post vaccination, H60-reactive memory cells were a stable population that comprised approximately 4-8% of splenic and peripheral blood CD8 cells. Approximately 50% of H60-tetramer+ cells had central memory phenotype, which could be ideal as central memory T cells mount strong anti-tumor responses. Similar results were obtained by using a prime/boost approach with H60-pulsed DCs. To test their function, CD44+ memory CD8 cells from H60-vaccinated mice were sort-purified and transferred into recipient B6 mice congenic for H60 (B6.H60; expression hematopoietically restricted ). By day 7 post transplant, H60-specific CD8+ TM from vaccinated mice comprised 70-90% of total splenic and blood CD8 cells, as compared to 1-5% in recipients of TM from unvaccinated mice. In contrast to anti-H60 responses by naïve CD8 cells, expansion of H60-tetramer+ cells from H60-vaccinated mice did not require CD4 help. To test whether target antigen distribution affects the re-expansion of H60-reactive cells from H60-vaccinated mice, expansion was compared in B6.H60 and B6.H60<right arrow>B6.actH60 (ubiquitous expression of H60 driven by an actin promoter) bone marrow chimeras. Approximately 60% of splenic and lymph node CD8 cells in both recipient groups were H60-tetramer+, though there was a trend towards increased overall numbers of tetramer+ cells in B6.H60 recipients. We are currently testing the GVL potency of TM from H60-vaccinated against mouse models of chronic phase (CP-CML) and blast crisis chronic myelogenous leukemia (BC-CML). In an ongoing experiment, very low numbers of CD8+ TM from H60-vaccinated mice mediate potent GVL against CP-CML relative to CD8+ TM from unvaccinated donors. Small numbers of CD8+ TM cells from vaccinated mice also mediated GVL against BC-CML, which is typically GVL resistant. Future studies will better define the potency of TM from H60-vaccinated mice and test these cells against CP-CML and BC-CML that do or do not express H60 but are otherwise identical. GVHD effect will also be tested. DisclosuresNo relevant conflicts of interest to declare.

Multivariate Statistical Analyses Demonstrate Unique Host Immune Responses to Single and Dual Lentiviral Infection

BackgroundFeline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) are recently identified lentiviruses that cause progressive immune decline and ultimately death in infected cats and humans. It is of great interest to understand how to prevent immune system collapse caused by these lentiviruses. We recently described that disease caused by a virulent FIV strain in cats can be attenuated if animals are first infected with a feline immunodeficiency virus derived from a wild cougar. The detailed temporal tracking of cat immunological parameters in response to two viral infections resulted in high-dimensional datasets containing variables that exhibit strong co-variation. Initial analyses of these complex data using univariate statistical techniques did not account for interactions among immunological response variables and therefore potentially obscured significant effects between infection state and immunological parameters.Methodology and Principal FindingsHere, we apply a suite of multivariate statistical tools, including Principal Component Analysis, MANOVA and Linear Discriminant Analysis, to temporal immunological data resulting from FIV superinfection in domestic cats. We investigated the co-variation among immunological responses, the differences in immune parameters among four groups of five cats each (uninfected, single and dual infected animals), and the “immune profiles” that discriminate among them over the first four weeks following superinfection. Dual infected cats mount an immune response by 24 days post superinfection that is characterized by elevated levels of CD8 and CD25 cells and increased expression of IL4 and IFNγ, and FAS. This profile discriminates dual infected cats from cats infected with FIV alone, which show high IL-10 and lower numbers of CD8 and CD25 cells.ConclusionsMultivariate statistical analyses demonstrate both the dynamic nature of the immune response to FIV single and dual infection and the development of a unique immunological profile in dual infected cats, which are protected from immune decline.

Intraepithelial Effector (CD3 +)/Regulatory (FoxP3 +) T-Cell Ratio Predicts a Clinical Outcome of Human Colon Carcinoma

Regulatory T cells (Tregs) express the forkhead box transcription factor (FoxP3) and suppress the antitumor immune response. We investigated whether the intratumoral densities of FoxP3(+) and effector CD3(+) lymphocytes are associated with prognosis of patients with colon cancer. FoxP3 and CD3 expression and location were determined in stage II and III colon carcinomas (n = 160) and normal mucosa (n = 25) by immunohistochemistry; CD4 and FoxP3 were localized by dual immunofluorescence microscopy. T-cell markers were compared with pathological variables, DNA mismatch repair status, and patient survival using Cox proportional hazards models. FoxP3(+) and CD3(+) T-cell densities were increased in carcinomas compared with autologous normal mucosa (P < .0001). An increase in intraepithelial FoxP3(+) cells was associated with poor tumor differentiation (P = .038), female sex (P = .028), and advanced patient age (P = .042). FoxP3(+) cell density was not prognostic, yet patients with tumors with reduced intraepithelial CD3(+) T-cell densities had reduced disease-free survival (DFS) rates (hazard ratio [HR], 1.87 [95% confidence interval, 1.10-3.16]; P = .018). A low intraepithelial CD3(+)/FoxP3(+) cell ratio predicted reduced DFS (46.2% vs 66.7% survival at 5 years; HR, 2.17 [95% confidence interval, 1.11-4.23]; P = .0205). The prognostic impact of these markers was maintained when tumors were stratified by mismatch repair status. By multivariate analysis, a low CD3(+)/FoxP3(+) cell ratio (P= .0318) and low numbers of CD3(+) T cells (P = .0397) predicted shorter DFS times and were stronger prognostic variables than tumor stage or number of lymph node metastases. A low intraepithelial CD3(+)/FoxP3(+) cell ratio and reduced numbers of CD3(+) T cells were associated with shorter patient survival time, indicating the importance of an effector to Treg cell ratio in colon cancer prognosis.

T Cell Infiltrate Predicts Long-Term Survival Following Resection of Colorectal Cancer Liver Metastases

While tumor infiltrating lymphocytes (TIL) have been shown to independently predict survival in primary colorectal cancer, the prognostic implications of TIL in resectable colorectal cancer liver metastases (CRCLM) have not been previously defined. This study examines the correlation between TIL numbers and survival following hepatic resection. We studied patients who survived <or=2 or >or=10 years following CRCLM resection. Immunohistochemistry was performed on tissue microarrays (TMAs) to determine the number of T cells within CRCLM. Correlation between TIL frequency and <or=2 or >or=10 year survival was determined while controlling for established prognostic factors. Of 162 patients, 104 survived <or=2 years and 58 survived >or=10 years. Independent correlates of 10-year survival following CRCLM resection included a high number of CD8 T cells, a low number of CD4 T cells, and a clinical risk score of <or=2 (P < 0.001). Among 10-year survivors, 31% of patients had a high number of CD8 T cells compared with 8% for <or=2 year survivors (P < 0.01). Surprisingly, only 22% of 10-year survivors had a high number of CD4 T cells, in contrast to 69% of those who died within 2 years (P < 0.001). The combination of CD8 and CD4 T cell counts was a more powerful predictor of survival than either marker alone. CRCLM T cell number is an independent correlate of long-term survival following liver resection. We conclude that CRCLM TIL analysis represents a potentially powerful prognostic tool which will require further validation prior to broad application.

Compartmentalization of Immune Responses in Human Tuberculosis: Few CD8+ Effector T Cells but Elevated Levels of FoxP3+ Regulatory T Cells in the Granulomatous Lesions

Immune responses were assessed at the single-cell level in lymph nodes from children with tuberculous lymphadenitis. Tuberculosis infection was associated with tissue remodeling of lymph nodes as well as altered cellular composition. Granulomas were significantly enriched with CD68+ macrophages expressing the M. tuberculosis complex-specific protein antigen MPT64 and inducible nitric oxide synthase. There was a significant increase in CD8+ cytolytic T cells surrounding the granuloma; however, CD8+ T cells expressed low levels of the cytolytic and antimicrobial effector molecules perforin and granulysin in the granulomatous lesions. Quantitative real-time mRNA analysis revealed that interferon-gamma, tumor necrosis factor-alpha, and interleukin-17 were not up-regulated in infected lymph nodes, but there was a significant induction of both transforming growth factor-beta and interleukin-13. In addition, granulomas contained an increased number of CD4+FoxP3+ T cells co-expressing the immunoregulatory cytotoxic T-lymphocyte antigen-4 and glucocorticoid-induced tumor necrosis factor receptor molecules. Low numbers of CD8+ T cells in the lesions correlated with high levels of transforming growth factor-beta and FoxP3+ regulatory T cells, suggesting active immunosuppression at the local infection site. Compartmentalization and skewing of the immune response toward a regulatory phenotype may result in an uncoordinated effector T-cell response that reduces granule-mediated killing of M. tuberculosis-infected cells and subsequent disease control.

Evaluation of immunological status in tumor-bearing dogs

The purpose of this study was to evaluate immunological status in dogs with cancers at different stages, in comparison with normal dogs. The population of canine peripheral blood lymphocytes (cPBL), lymphocyte phenotypes, interleukin (IL)-6 activity and alpha 1-acid glycoprotein (α 1-AG) level were analyzed. The tumor-bearing dogs had higher numbers of leukocytes than normal dogs, the count being higher in dogs with more advanced tumors. In the tumor-bearing dogs, differential leukocyte counts revealed higher percentages of inflammatory cells such as neutrophils, acidophils and monocytes, and lower numbers of CD4 +T cells, than in normal dogs, the lymphocyte counts becoming much lower with tumor progression. In the tumor-bearing dogs, the CD8 +T cell count at the early tumor stage was similar to that in normal dogs, but decreased with tumor progression, possibly reflecting the development of humoral immunity (Th2). Plasma IL-6 and TGF-β activities were high in the tumor-bearing dogs. The plasma α 1-AG concentration was also significantly high in the tumor-bearing dogs. Our findings suggest that assay of IL-6, TGF-β and α 1-AG may be very useful for prognostication in dogs with cancer, and that anti-tumor immunity is potently suppressed in such dogs.