Thymus-dependent Lymphocytes Research Articles

Comparative histological changes in the structure of the spleen and kidneys of experimental chickens exposed to the action of “Guidamis” as an adjuvant for vaccination against infectious bronchitis

The article provides a histological description of the internal organs – the spleen and kidneys of chickens exposed to the action of “Guidamis” as an adjuvant. A lysate was tested as part of a sample of the vaccine PB-07 against infectious bronchitis developed by researchers of the Department of Virology and Tumor Diseases of Birds of the Russian Agricultural Academy (Lomonosov). The experiments were carried out on Lohmann Brown chickens in the conditions of the Laishev branch of “Ak Bars” Poultry Company, the Republic of Tatarstan. The prolonged action of the vaccine against infectious bronchitis and the use of “Guidamis” as an adjuvant was manifested as an increase in the proliferation and differentiation of lymphoid tissues in the numerous lymph nodes. The use of the vaccine and the adjuvant had a weak reactogenic effect. In the spleen of control chickens, hyperplasia of lymphoid tissue cells is weakly expressed. In the periarterial zone of nodules of the thymus-dependent lymphocytes, the cell density is low. In the kidneys, for fourteen days after the vaccination against infectious bronchitis using Guidamis, the moderate granular dystrophy of the epithelium, mainly of individual distal tubules, and local moderate manifestations of edema of the capsule of the renal corpuscles were observed. In the kidneys, signs of pronounced edema of the capsule cavity, significant disturbances in the patency of the tubular network caused by swelling and partial desquamation of the epithelium, as well as the presence of epithelium and protein mass in the lumen of the tubules, were observed.

Loss of CD31 receptor in CD4+ and CD8+ T-cell subsets in children with primary hypertension is associated with hypertension severity and hypertensive target organ damage.

Primary hypertension is associated with still poorly known T-cell dependent immunity defects that participate in the disease development. However, the relationship between peripheral T-cell subset distribution and disease severity in humans is not known. The aim of the study was to find out if target organ damage in adolescents with primary hypertension is associated with thymus-dependent lymphocytes renewal reflected by changes in the T-cell subset phenotype characteristics. Using seven-color flow cytometry technique, we assessed CD31, CCR7 and CD28 receptors expression in CD45RA and CD45RO bearing peripheral CD4 and CD8 T-cell subsets. The study included 32 hypertensive children/adolescents and 35 sex-matched and age-matched controls. Children with primary hypertension had slightly increased CD4 T-cell pool but decreased population of CD31 expressing CD4 T-cell subsets (recent thymic emigrants). Frequency of the CD4 and CD4/CD45RA+ T cells lacking CD31 correlated positively with the hypertensive organ damage markers (pulse wave velocity, central blood pressure, left ventricular mass index). Left ventricular hypertrophy was associated with decreased CD4/CD45RA:CD4/CD45RO ratio, loss of the CD31 receptor in the CD4 and CD8 T-cell subsets and increased population of effector/memory T cells bearing CD8/CD28 and CD8/CD45RA+/CCR7 phenotype. Regression analysis revealed that these associations were independent of age, sex, and BMI. The results suggest that subclinical arterial injury and left ventricular hypertrophy in adolescents with primary hypertension is associated with declined thymic function and increased pool of T cells bearing effector/memory phenotype.

Research about NG-PDT Influence to Thymus-Dependent Lymphocyte Subpopulation from Peripheral Blood of Bronchogenic Carcinoma

Research about NG-PDT Influence to Thymus-Dependent Lymphocyte Subpopulation from Peripheral Blood of Bronchogenic Carcinoma

Effects of astrogaloside on the inflammation and immunity of renal failure patients receiving maintenance dialysis.

Chronic renal failure is a type of clinical syndrome originating from chronic renal diseases. The aim of the study was to investigate the effect of astrogaloside on the inflammation and immunity of renal failure patients receiving maintenance dialysis. We randomly selected 92 renal failure patients receiving maintenance dialysis who were admitted to hospital for treatment between May, 2015 and April, 2016. Patients were randomly divided into the control (n=46) and observation (n=46) groups. Patients in the control group received the regular dialysis plus the basic treatment in Western medicine, while in the observation group, patients additionally received astrogaloside via intravenous injection as treatment. We compared the clinical efficacy of patients between the two groups, residual renal function (RRF), changes in urine volume, variations in inflammatory indicators [C-reaction protein (CRP), interleukin-6 (IL-6), IL-17, and tumor necrosis factor-α (TNF-α)] before and after treatment, and the levels of the thymus-dependent lymphocyte (T cells) subgroup (CD3+, CD4+, CD8+ and CD4+/CD8+) in the immune system of patients after treatment. In the observation group, the total effective rate was significantly higher than that in the control group (P<0.05). After 6 months, RRF and the urine volume of patients in the two groups were decreased when compared with the levels before treatment, and the decreasing rates of RRF and urine volume in the observation group were significantly lower than those in the control group (P<0.05). After treatment, the levels of human serum C reaction protein (hs-CRP), IL-6, IL-17 and TNF-α in the two groups were lower than those before treatment, and the decrease in the observation group was more significant than that in the control group (P<0.05). Following treatment, the levels of CD3+, CD4+ and CD4+/CD8+ in the observation group were higher than those in the control group, and the level of CD8+ was lower than that in the control group (P<0.05). In conclusion, astrogaloside can delay the decrease in RRF of renal failure patients receiving the maintenance dialysis, ameliorate the inflammatory responses, and enhance the immune function, thereby increasing the disease resistance of patients and improving the clinical symptoms.

On the prenatal initiation of T cell development in the opossum Monodelphis domestica.

Thymus-dependent lymphocytes (T cells) are a critical cell lineage in the adaptive immune system of all jawed vertebrates. In eutherian mammals the initiation of T cell development takes place prenatally and the offspring of many species are born relatively immuno-competent. Marsupials, in contrast, are born in a comparatively altricial state and with a less well developed immune system. As such, marsupials are valuable models for studying the peri- and postnatal initiation of immune system development in mammals. Previous results supported a lack of prenatal T cell development in a variety of marsupial species. In the gray short-tailed opossum, Monodelphis domestica, however, there was evidence that αβT cells were present on postnatal day 1 and likely initiated development prenatally. Demonstrated here is the presence of CD3ε+ lymphocytes in late-stage embryos at a site in the upper thoracic cavity, the site of an early developing thymus. CD3ε+ cells were evident as early as 48h prior to parturition. In day 14 embryos, where there is clear organogenesis, CD3ε+ cells were only found at the site of the early thymus, consistent with no extra-thymic sites of T cell development in the opossum. These observations are the first evidence of prenatal T cell lineage commitment in any marsupial.

Chinese Herbal Extractions for Relieving Radiation Induced Lung Injury: A Systematic Review and Meta-Analysis.

Background. Radiation induced lung injury (RILI) is one of the most common and severe side effects of thoracic radiotherapy. In this meta-analysis, the effects of Chinese herbal extractions (CHE) for preventing and treating RILI are evaluated. Methods. Randomized Controlled Trials (RCTs) from five databases were identified. Studies were evaluated and the relevant data were extracted by two authors independently. Differences were resolved by a third party. Meta-analysis was conducted using RevMan 5.0. Results. In total, 2734 participants receiving thoracic radiotherapy were included in 28 RCTs, and 16 CHE were evaluated. Meta-analysis showed that CHE intervention significantly reduced the incidence of acute radiation pneumonitis (RP) and radiation induced pulmonary fibrosis (RIPF). In CHE group, total effective rate and remission rate of RILI patients were significantly higher. Patient's quality of life (Qol) and clinical symptoms and signs were improved significantly. Inflammatory cytokines decreased, and thymus dependent lymphocytes subgroups were improved. Conclusion. CHE intervention may have clinical effectiveness for relieving RILI and related symptoms and signs and lead to improvement of Qol. However, more double-blind, multicenter, large-scale RCTs are needed to support this theory. Trial Registration. PROSPERO International prospective register of systematic reviews has registration number CRD42016043538.

Leukemia: Derived heat shock protein gp96-peptide complex contribution to T cell and dendritic cell activation

The molecular chaperone, heat shock protein gp96 (HSP-gp96), has been shown to have roles in the synthesis, processing and transport of tumor antigens. Therefore, the capacity for HSP-gp96 to induce dendritic cells (DCs), thymus-dependent lymphocytes (T lymphocytes), natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) was investigated. Recombinant adenovirus (AD) containing HSP-gp96 (AD-gp96), as well as gp96-peptide complex from the human leukemia cell lines, K562, HL-60 and U937, was prepared. Purified gp96-peptide complex was found to stimulate the proliferation of T lymphocytes, increase the activity of NK cells and CTLs and induce the secretion of cytokines, compared with ADgp96. In the latter case, levels of IFN-γ and TNF-α were found to increase and levels of IL-12(P70) and IL- 10 decreased. In combination, these results indicated that the gp96-peptide complexes derived from the tumor cells contributed to the activation of lymphocytes and increase the presentation of tumor antigen. Furthermore, the chaperone function of gp96 promoted the maturation of DCs, enhanced the antigen presention function of DCs and induced the secretion of cytokines by DCs. Therefore, gp96-peptide complex derived from the tumor cells potentially represents an immunization therapy for the elimination of residual leukemia cells. Key words: Leukemia, heat shock protein gp96, dendritic cells, cytotoxic T lymphocytes.

Effects of low dose of hydrocortisone on circulating thymus-dependent lymphocyte apoptosis in patients with septic shock

Objective To investigate the effects of low-dose of hydrocortisone on circulating thymus-dependent lymphocyte (T lymphocyte) apoptosis in patients with septic shock. Method fifty-seven patients with septic shock admitted into ICU from January 2006 to January 2009 were prospectively randomized (random number) to treatment group and control group. Another 20 healthy volunteers and 18 patients with sepsis alone were included as external control groups.The patients of treatment group and control group were treated with low-dose of hydrocortisone and placebo,respectively. Samples of peripheral blood were taken from healthy volunteers and patients 0 hr,24 hrs,48 hrs,72 hrs and 168 hrs after onset of the disease to determine the circulating T lymphocyte apoptosis by using theassays of Annexin V and flow cytometry. Least significant difference t -test was used for multiple comparisons. Results The percentage of Annexin V-positive CD4+ T lymphocytes in the primary stage was (11.01 +4.52)% in septic shock patients, (4.41 + 1.45)% in healthy volunteers, and (7.87 + 3. 82)% in patients with sepsis alone. And in the initial setting, the percentage of Annexin V-positive CD4+ T lymphocytes in the septic shock patients was higher than that in healthy volunteers ( P 0.05 between three groups). The percentages of Annexin V-positive CD4+ T lymphocytes in control group after 24 his, 48 hrs and 72 hrs were(13.51+6.85)%, (19.39 + 6.63)% and (15.33+ 6.21)%, respectively. And the percentages of Annexin V-positive CD4+ T lymphocytes in treatment patients after 24 hrs, 48 hrs and 72 hrs were (17.4 + 7.21)%, (22.61 + 5.64)%, and (25.73 + 6.91)%, respectively. The percentage of Annexin V-positive CD4+ T lymphocytes in septic shock patients was higher than that in control groups ( P 0.05 between two groups). In the meantime, the percentages of Annexin V-positive CDS'1' T lymphocytes in control group and in treatment group were (12.72+ 19.69)% and (13.88 + 13.28)%, respectively (P >0.05). Conclusions Low-dose of hydrocortisone could induce CD4+ T lymphocyte apoptosis and has no effects on CD8+ T lymphocyte apoptosis when it is used to treat septic shock. Key words: Septic shock; Lymphocyte; Apoptosis; Hydrocortisone

Differential Roles of Costimulatory Signaling Pathways in Type 1 Diabetes Mellitus

Insulin-dependent type 1 diabetes mellitus (T1DM) is a chronic immune mediated disease in which T lymphocytes recognizing pancreatic islet cell antigens play a major role in the disease process [2, 3]. It has become increasingly clear that autoreactive T cells are important in the development and progression of this disease. The initial event in the pathogenesis of autoimmune disease is thought to be the priming of naive autoreactive T cells, perhaps as a consequence of environmental exposure to shared epitopes (antigenic mimicry) or generalized inflammation/innate immunity [2-4]. Maximum activation of naive T lymphocytes requires at least two independent signals. The first, an antigen-specific signal is sent via the clonotypic T cell receptor (TCR) which recognizes the appropriate peptide-MHC complex. The second signal, termed costimulatory signal, is independent of the antigen receptor and is critical to allow full activation. In addition, costimulatory signaling controls proliferation, prevents anergy and/or apoptosis, and induces differentiation to effector function and memory status. Costimulatory signals also regulate inhibitory receptors upon lymphocyte activation in a time-dependent manner [5, 6]. It is now clear that the delicate balance of these positive and negative regulatory pathways underpins the outcome - self-tolerance versus autoimmunity [7]. Therefore, the immune response observed in T1DM seems to be related to an imbalance of the sensitively regulated process which usually controls the activation of thymus-dependent lymphocytes. In this article, we summarize the role of costimulation in the development and progression of T1DM. In addition to the basic biology, we highlight important clinical advances in costimulation blockade that may impact this disease.

Use of basiliximab and daclizumab in kidney transplantation

Kidney transplantation represents a major medical victory in patients with whom dialysis and medical therapy have failed. To increase survival rates and optimize the use of limited organs, both patient care and immunosuppression therapy must be improved. Reduction in rejection episodes or severity of rejection may ultimately improve long-term allograft survival. Traditional engineered monoclonal antibodies have been associated with severe cytokine release reactions and an increased risk of opportunistic infections. Basiliximab and daclizumab are chimeric and humanized monoclonal antibodies which inhibit thymus-dependent lymphocyte proliferation. Interleukin-2 also affects the proliferation of natural killer cells, macrophages and monocytes, bursa-equivalent lymphocytes, epidermal dendritic cells, and lymphokine-activated killer cells. Interleukin-2 receptor antagonists have been shown to reduce the incidence of acute rejection without increasing the incidence of opportunistic infections or malignancy. Further studies are needed to evaluate the overall effect of these agents on long-term patient and allograft survival.

Identical T cell clones are located within the mouse gut epithelium and lamina propia and circulate in the thoracic duct lymph.

Murine gut intraepithelial (IEL) T cell receptor (TCR)-alpha/beta lymphocytes bearing CD8alpha/13 or CD8alpha/alpha coreceptors have been shown previously to express different oligoclonal TCR beta chain repertoires in the same mouse, in agreement with other evidence indicating that these two populations belong to different ontogenic lineages, with only CD8alpha/beta+ IELs being fully thymus dependent. CD8alpha/beta+, but not CD8alpha/alpha+, T lymphocytes are also present in the lamina propria. Here, we show that CD8alpha/beta+ lymphocytes from the lamina propria and the epithelium are both oligoclonal, and that they share the same TCR-beta clonotypes in the same mouse, as is also the case for CD4alpha T cells. Furthermore, identical T cell clones were detected among CD8alpha/beta IELs and CD8alpha/beta+ blasts circulating into the thoracic duct (TD) lymph of the same mouse, whereas TD small lymphocytes are polyclonal. These findings must be considered in light of previous observations showing that T blasts, but not small T lymphocytes, circulating in the TD lymph have the capacity of homing into the gut epithelium and lamina propria. These combined observations have interesting implications for our understanding of the recirculation of gut thymus-dependent lymphocytes and their precursors, and of the events leading up to the selection of their restricted TCR repertoire.

Regulation of mouse immuno-responses by a natural suppressor cell clone from bone marrow

Natural suppressor (NS) activity is mediated by several kinds of cell lineages in bone marrow. We demonstrated that a NS cell, clone 7-31, was obtained after limiting dilution with bone marrow culture supplemented with WEHI supernatant. Clone 7-31, was capable of non-specific suppression of the generation of cytotoxic T lymphocytes without major histocompatibility complex restriction. Suppression of cytotoxic thymus-dependent lymphocyte (CTL) generation was also induced with a cell-free supernatant from the 7-31 cells. Interleukin-2 (IL-2) containing concanavallin-A-conditioned medium could not reverse the suppression. The supernatant did not inhibit the proliferation of IL-2-dependent CTLL-2 cells and rapidly growing tumour cells and fibroblasts. Thus, this bone marrow suppressor cell produces a soluble factor that inhibits the generation of CTL in vitro, and prolongs the acceptance of skin allografts in in vivo.

Number and Percentage of NK-Cells Are Decreased in Growth Hormone-Deficient Adults

Snell-Bagg mice and Ames dwarf mice repeatedly show severe immunodeficiencies, affecting mostly the thymus-dependent lymphocyte system, probably caused by growth hormone deficiency. In growth hormone-deficient children contradictory data on the immune status have been reported. We investigated indices of cellular immunity in 22 adult patients with proven growth hormone deficiency in comparison to those in 100 healthy volunteers. Cellular immunity was assessed using total leukocyte count, percentage lymphocytes, and percentage and absolute numbers of CD3, CD4, CD8, CD19, and CD3−CD56+(NK)-cells. Comparison revealed statistically significant lower percentage and absolute number of NK-cells (P< 0.001). Except for a trend toward an increased CD4/CD8 ratio, no statistically significant differences for B- and T-lymphocytes could be obsereved. No correlation between the percentage and absolute number of NK-cells, on one hand, and the duration of growth hormone deficiency or prolactin level, on the other hand, could be demonstrated. In all these respects men did not differ from women. So, in growth hormone-deficienct adults the percentage and absolute number of NK-cells are decreased.

Synthesis of a new biological response modifier thyrnosin β4 analogue and its restorative effect on depressed

Thymosin β4 is a polypeptide isolated from thymosin fraction 5. This peptide exhibits important activities in the regulation and differentiation of thymus-dependent lymphocytes. An analogue of thymosin β4, [Phe(4F)12] deacetyl- thymosin β4, was synthesized by a solution method, followed by deprotection with 1 M trifluoromethanesulphonic acid (TFMSA)-thioanisole (molar ratio, 1:1) in trifluoroacetic acid (TFA) in the presence of dimethlselenium. Finally, the deprotected peptide was incubated with dithiothreitol to reduce sulphoxide on the methionine side chain. The synthetic [Phe(4F)12]deacetyl-thymosin β4 was found to have a restoring effect on the impaired blastogenic response of T-lymphocytes isolated from uraemic patients with recurrent infectious diseases. This analogue exhibited stronger restorative activity than that of our synthetic deacetyl-thymosin β4.

Inhibition of lymphocyte proliferation by amantadine and its isomer, 2-aminoadamantane; impact on Lyt-2+ T cells while sparing L3T4+ cells

The present in vitro investigations on amantadine (AmTd) and its isomer 2-aminoadamantane (2-NH2-Adam), and the corresponding analogs, 1-nitroadamantane (1-NO2-Adam) and 2-nitroadamantane (2-NO2-Adam), were undertaken to gain information about molecular features that might have a dominant role in inhibiting T lymphocyte proliferation and to determine whether all, or a subpopulation of thymic-dependent (T) lymphocytes might be impacted by these drugs. Studies were done using lymphocytes from untreated normal mice as well as cloned murine cytotoxic T lymphocytes, CTLL cells. T lymphocytes were defined by their proliferative response to concanavalin A (Con A), and thymic-independent (B) lymphocytes by their proliferative response lipopolysaccharide (LPS). Proliferation of CTLL cells was induced by supplementing the culture medium with lymphokine-containing medium or by adding recombinant interleukin (IL)-2. Proliferation was assessed by quantifying cellular incorporation of tritiated thymidine. The data show that the aminoadamantanes, AmTd and 2-NH2-Adam, impacted on Lyt-2+ T lymphocytes while sparing L3T4+ T lymphocytes. In addition, the data show that the location of the substituent group on the adamantane ring altered the molecule's capacity to modulate lymphocyte activity. And finally, results of studies comparing the inhibitory activity of AmTd and 1-NO2-Adam suggest a non-lysosomal mechanism of action. Possible implications of these findings are discussed.

Erythrocyte receptor (CD2)-bearing T lymphocytes are affected by diet in experimental pulmonary tuberculosis

Outbred (Hartley) and inbred (strain 2 and 13) guinea pigs were vaccinated with Mycobacterium bovis BCG and placed on isocaloric, purified diets containing either 10 or 30% ovalbumin or on commercial guinea pig chow. Six weeks later, the animals were challenged by the respiratory route with virulent M. tuberculosis H37Rv. At intervals postchallenge, groups were tuberculin tested and sacrificed. Thymus-dependent (T) lymphocytes were enumerated either by rosette formation with rabbit erythrocytes or by the indirect immunofluorescence assay (IFA) with a guinea pig pan-T-cell monoclonal antibody, 8BE6. Protein-deficient guinea pigs of all three strains had significantly fewer erythrocyte rosette-forming (CD2+) T cells in the peripheral blood, and malnourished strain 2 and Hartley guinea pigs exhibited reduced levels of CD2+ T cells in the thymus. In contrast, animals of all three strains fed the low-protein diet harbored more CD2+ T cells in the bronchotracheal lymph nodes than did their control-fed counterparts. A larger proportion of lymphocytes from the blood and lymph nodes of all three strains were IFA positive than formed erythrocyte rosettes regardless of diet treatment. Diet had no effect on IFA-positive lymphocytes in those organs. Protein deficiency is associated with significant alterations in the number and/or distribution of T lymphocytes expressing functional CD2-receptors in BCG-vaccinated animals exposed to virulent mycobacteria by the pulmonary route. These alterations may contribute to the reduction in BCG vaccine efficacy observed in this model.

Therapy of secondary T-cell immunodeficiencies with biological substances and drugs.

Thymus-dependent (T) lymphocyte defects are common in cancer. Recent advances in the understanding of the regulation of T-cell development by biologicals and drugs now allow the formulation of better strategies of immune reconstitution to correct these defects. Thymic hormone preparations of several types offer one type of approach; however, they are somewhat limited in their reconstitutive ability. Interleukins (IL), particularly IL-1 and IL-2, appear to be complementary to the actions of thymic hormones in promoting T-cell development. Two classes of thymomimetic drugs have been identified and are represented by levamisole and isoprinosine. These drugs mimic by indirect and direct actions, respectively, the actions of thymic hormones. Newer analogs of these compounds have emerged which appear more effective. Also new factors, e.g. pituitary factors, are emerging which may be potent regulators of the immune system and useful in therapy. These agents may now be more effectively integrated with cytodestructive therapy in cancer treatment.

Are The Immune Responses to Endocrine Autoantigens Genetically Restricted?

Thymus-dependent (T) lymphocytes predominantly recognize antigens only when associated with products of the major histocompatibility complexes (MHC). This phenomenon is known as “genetic restriction” of the immune response. MHC Class II antigens restrict immunoregulatory T cells whereas Class I molecules determine the recognition by cytotoxic T cells or tumor cells and virus-infected cells. Genetic variations in cell-mediated and humoral responses to antigens have been related to variations in the first extracellular domain of Class II molecules.1 The bulk of data relate to the mouse, but similar information relative to the human is rapidly emerging.

Nutritional modulation of intestinal mucosal immunity.

Protein-energy malnutrition results in an increased risk of gastrointestinal infection. This can be attributed in part to impaired immune responses. Cell-mediated immunity is decreased as judged by reduced number and function of thymus-dependent lymphocytes, impaired delayed cutaneous hypersensitivity reactions, and decreased production of lymphokines. Concentration of secretory IgA is reduced and there are fewer intraepithelial lymphocytes. Antibody responses following viral vaccine administration are reduced and there is decrease in natural killer cell activity. In addition, the number of bacteria binding to epithelial cells is increased. These changes are observed also in certain selected nutrient deficiencies, such as that of vitamin A. It is suggested that impaired systemic and mucosal immunity contributes to the increased frequency and severity of intestinal infections seen in undernourished individuals.

Low-Dose X-Irradiation and 4-Hydroperoxycyclophosphamide Distinguishes Three LYT-1+ Lymph Node T-Cell Subtypes That Respond to Specific Antigen In Vitro

We investigated whether preculture exposure to low-dose X-irradiation and culture treatment with 4-hydroperoxycyclophosphamide (4-HPCY), a synthetic derivative of cyclophosphamide (CY), might distinguish subtypes of thymic-dependent (T) lymphocytes that respond to specific antigen in vitro. Lymph node (LN) cells were obtained from mice pretreated with CY and immunized with aggregated (A) human IgG (HGG) in Freund's complete adjuvant (CFA), and proliferation was assessed by incorporation of tritiated thymidine. Primed LN cells were untreated or exposed to low-dose irradiation before being cultured in medium alone and in medium containing 4-HPCY. The results show that these agents (irradiation and 4-HPCY) distinguished, in a dose-dependent manner, subtypes of T-cells which contribute to the specific antigen-stimulated proliferative response in vitro. For LN T-cells and LN Lyt-1+ T-cells, 20-25 rads and 1.0 microM 4-HPCY inactivated non-overlapping cell subtypes that respectively accounted for 26% and 28% of the response to HGG. The remaining 46% of HGG-responding cells were not affected by either agent. Although similar cell subtypes were discerned in unseparated LN cells, it required use of higher agent-doses. Cell cycle analysis revealed that treatment with irradiation, 4-HPCY, and the combination (both agents) caused S-phase arrest of 29%, 30%, and 55% of HGG-responding cells, respectively. Thus, identification of these cell subtypes could not be attributed to agent-mediated inactivation of HGG-responding cells that might be in exclusively different phases of the cell cycle.