The complex interactions between cancer and host cells are far from being fully elucidated. Assessment of
Th1/Th2/Th3/Tr1 balance is an interesting approach to explain immunological disturbances in lymphomas. The aim of our
study was to assess mRNA for pro- and anti-inflammatory cytokines in T-cells in 20 children with Hodgkin- and non-Hodgkin
lymphomas. CD4+ and CD8+ cells were isolated from whole peripheral blood and four different cytokine mRNA levels (IFN-&#947;,
IL-10, IL-4, TGF-&#946;) were determined by real-time PCR technique. Comparing to the control group, we found lower expression
of mRNA for IFN-gamma in CD4+ cells at the time of lymphoma diagnosis. It may be one of the pathogenetic mechanisms of
impaired immunity in these patients

Chyczewski, Lech

Kovalchuk, Oksana

Kowalczyk, Jerzy

Krawczuk-Rybak, Maryna

Malinowska, Iwona

Matysiak, Michał

Mitura-Lesiuk, Małgorzata

Łuczyński, Włodzimierz

Folia Histochemica et Cytobiologica

English

The complex interactions between cancer and host cells are far from being fully elucidated. Assessment of Th&lt;sub&gt;1&lt;/sub&gt;/Th&lt;sub&gt;2&lt;/sub&gt;/Th&lt;sub&gt;3&lt;/sub&gt;/Tr&lt;sub&gt;1&lt;/sub&gt; balance is an interesting approach to explain immunological disturbances in lymphomas. The aim of our study was to assess mRNA for pro- and anti-inflammatory cytokines in T-cells in 20 children with Hodgkin- and non-Hodgkin lymphomas. CD4&lt;sup&gt;+&lt;/sup&gt; and CD8&lt;sup&gt;+&lt;/sup&gt; cells were isolated from whole peripheral blood and four different cytokine mRNA levels (IFN-&amp;#947;, IL-10, IL-4, TGF-&amp;#946;) were determined by real-time PCR technique. Comparing to the control group, we found lower expression of mRNA for IFN-gamma in CD4&lt;sup&gt;+&lt;/sup&gt; cells at the time of lymphoma diagnosis. It may be one of the pathogenetic mechanisms of impaired immunity in these patients

Michał Matysiak

Jerzy Kowalczyk

Lech Chyczewski

Iwona Malinowska

Małgorzata Mitura-Lesiuk

Maryna Krawczuk-Rybak

Oksana Kovalchuk

Włodzimierz Łuczyński

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Lower expression of mRNA for interferon-gamma in T helper cells of children with newly diagnosed lymphomas

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FOLIA HISTOCHEMICAET CYTOBIOLOGICAVol. 43, No. 3, 2005pp. 169-171Lower expression of mRNA for interferon-gamma in T helper cells of children with newly diagnosed lymphomasWłodzimierz Łuczyn´ski1, Oksana Kovalchuk4, Maryna Krawczuk-Rybak1,Małgorzata Mitura-Lesiuk2, Iwona Malinowska3, Lech Chyczewski4, JerzyKowalczyk2 and Michał Matysiak31Department of Pediatric Hematology and Oncology, Medical University, Białystok;2Department of Pediatric Hematology and Oncology, Medical University, Lublin;3Department of Pediatric Hematology and Oncology, Medical University, Warszawa;4Department of Clinical Molecular Biology, Medical University, Białystok, PolandAbstract: The complex interactions between cancer and host cells are far from being fully elucidated. Assessment ofTh1/Th2/Th3/Tr1 balance is an interesting approach to explain immunological disturbances in lymphomas.  The aim of ourstudy was to assess mRNA for pro- and anti-inflammatory cytokines in T-cells in 20 children with Hodgkin- and non-Hodgkinlymphomas. CD4+ and CD8+ cells were isolated from whole peripheral blood and four different cytokine mRNA levels (IFN-γ,IL-10, IL-4, TGF-β) were determined by real-time PCR technique. Comparing to the control group, we found lower expressionof mRNA for IFN-gamma in CD4+ cells at the time of lymphoma diagnosis. It may be one of the pathogenetic mechanisms ofimpaired immunity in these patients.Key words: Lymphoma - Children - T-cells - Interferon-gamma - ImmunosuppressionIntroductionLeukemias and lymphomas are the most frequent neo-plasmatic diseases in children. The complex interactionsbetween cancer and host cells are far from being fullyelucidated. One of the approaches to understand theserelationships can be assessment of cytokines producedby both neoplasmatic and host cells. We, as well as theother authors, showed anti-inflammatory cytokinepredominance (Th2, Th3, Tr1 subpopulations of T-cells)at protein and mRNA levels at the time of leukemiadiagnosis in children [5, 7, 12]. Data concerningTh1/Th2/Th3/Tr1 balance in lymphomas are not veryclear. The aim of our study was to determine pro- andanti-inflammatory cytokine mRNA expression profilesin T cells of children with Hodgkin- and non-Hodgkinlymphomas. Materials and methodsTen patients with Hodgkin lymphoma (HL) and 10 with non-Hodg-kin lymphoma (NHL) were prospectively enrolled in this study.Peripheral blood (1 ml) was taken at the time of diagnosis before thetreatment. Control group included 30 children from the Departmentof Paediatric Surgery subjected to minor surgical operations. Thestudy was approved by the Ethics Comitee of the Medical Universityof Białystok. Informed consent was obtained for collecting thematerials for this study. CD4+ and CD8+ cells were isolated from whole peripheral bloodby immunomagnetic bead separation (Dynabeads, Dynal Biotech,Oslo, Norway). mRNA was isolated from sorted lymphocyte sub-populations using Dynabeads mRNA Direct Micro Kit  (Dynal)according to the producer instructions. First strand cDNA was syn-thesized using random hexamers as primer and High Capacity cDNAArchive Kit by Applied Biosystems. Four different cytokine mRNAslevel (IFN-γ, IL-10, IL-4, TGF-β1) were determined by  real-timePCR technique with the TaqMan chemistry using ready-to-usedAssays-on-Demand Gene Expression Products by AppliedBiosys-tems which contain target-specific primers and probe and TaqManUniversal Master Mix, containing AmpErase uracil-N-glycosylase(UNG) to prevent the re-amplification of carryover PCR products.The PCR amplification and fluorescence data collection were per-formed with ABI PRISM 7900HT Sequence Detection System (Ap-plied Biosystems). To normalize the amount of expressed cytokinemRNAs, the internal housekeping gene GAPDH was used and eachcomplementary DNA (cDNA) product was tested in triplex for eachCorrespondence: W. Łuczyn´ski, Dept. Pediatric Oncology, Medi-cal University, Waszyngtona 17, 15-274 Białystok, Poland; e-mail:vlodek@amb.edu.plof four cytokines mRNA and GAPDH mRNA. To calculate our datawe used comparative Ct  method for relative quantification (ΔΔCtmethod) which describes the change in expression of the target genein a test sample relative to a calibrator sample and provides accuratecomparison between the initial level of template in each sample. Asa calibrator sample we used Total Raji RNA (Applied Biosystems)which was processed in the same way as the test samples. Data wereanalyzed with Sequence Detector System (SDS) software version 2.1(Applied Biosystems). Significance levels were calculated according to the nonparame-tric Mann-Whitney U-test (difference between the lymphoma andthe control group) and the Spearman correlation coefficient (corre-lation between mRNAs for the examined cytokines). Level of p<0.05was regarded as significant. Results and discussionWe found lower mRNA levels for IFN-γ in CD4+ cellsin children with newly diagnosed lymphomas in com-parison to the control group (-ΔΔCt: 10.02 vs. 8.52,p=0.03). There were no statistically significant differen-ces in mRNA levels between the control and the lym-phoma group for the other assessed cytokines, i.e. IL-10,IL-4 and TGF-β1 (p>0.05, Table 1).In the lymphoma and control groups (analysed sep-arately and together), we noted positive correlationsbetween mRNA levels for all the assessed cytokines inCD4+ and CD8+ cells (e.g. IFN-γ and IL-10, IL-4, TGF-β,r=0.71, r=0.61, r=0.60, respectively).These preliminary results demonstrate that levels ofexpression for IFN-γ mRNA in T helper cells are lowerin children with lymphomas than in healthy children.Similarly, IFN-γ production in peripheral blood mono-nuclear cells was at low levels in Helicobacter pylori-induced low-grade gastric mucosa-associated lymphoidtissue (MALT) lymphoma [11]. However, opposite re-sults were obtained by Gergely et al. [2] - they foundincreased percentage of IFN-γ-producing T cells (Th1)in untreated lymphoma cases. Their study was perfomedat protein level (flow cytometry) and our study - atmRNA level. This may be one of possible explanationsfor different results. Interestingly, Mori et al. [9] foundincreased percentages of Th1 and Th2 cells in patientswith untreated B-cell diffuse large cell lymphoma butthe mean Th1/Th2 ratio was lower than in patients inremission - is it a sign of IFN-γ deficiency? The authorssuggest that Th1/Th2 imbalance could play a significantrole in lymphoma genesis and durable remission.Some authors postulate a relationship between typeof immune response and prognosis in lymphomas:tumor-resistant hosts developed Th1 dominant response,whereas susceptible hosts - Th2 dominant response [4].Moreover, in cases positive for Th1 and Th2 profilechemokines (i.e. ST2(L), CCR5 and CXCR3 express-ion), favourable prognosis was shown in comparison tocases negative for Th1 or Th2 profile [10]. We have not noted any differences in mRNA levelsfor IL-10, IL-4 and TGF-β between the control andlymphoma group. In Hodgkin disease, Mainou-Fowleret al. [8] observed increased production of IL-4 by CD8+cells from peripheral blood. Kaizu et al. [3] reported"disruption of the cytokine network", i.e. increase inIFN-γ and IL-10 after splenectomy in aggressive NK cellleukemia/lymphoma. Correlations between all the as-sessed mRNAs found in our experiment suggest theirinterdependence in cytokine network in the human or-ganism. Results of our investigation could be useful in thedevelopment of new therapeutic strategies using Th1cytokines. First reports in this field demonstrate induc-tion of humoral immune response after adenovirus-mediated intralesional interferon-gamma gene transfer[1].Acknowledgements: The authors thank Iwona Buchowiec, Graz˙ynaDenisiuk and Agnieszka Kryszpiniuk for their excellent technicalsupport. References[ 1] Dummer R, Hassel JC, Fellenberg F, Eichmuller S, Maier T,Slos P, Acres B, Bleuzen P, Bataille V, Squiban P, Burg G,Urosevic M (2004) Adenovirus-mediated intralesional interfe-ron-gamma gene transfer induced tumor regressions in cuta-neous lymphomas. Blood 104: 1631-1638[ 2] Gergely L, Aleksza M, Varoczy L, Ponyi A, Sipka S, Illes A,Szegedi G (2004) Intracellular IL-4/IFN-gamma producing pe-ripheral T-lymphocyte subsets in B cell non-Hodgkin’s lym-phoma patients. Eur J Haematol 72: 336-341[ 3] Kaizu K, Maeda M, Ohkawa T, Hayashida M, Nakajima S,Sugisaki Y, Fukunaga Y (2004) Marked elevation of soluble fasligand and cytokine secretion after splenectomy in aggressiveTable 1. Cytokine mRNA expression revealed by real-time PCR (ΔΔCt method) in CD4 and CD8 cells of children with lymphoma (medianvalues)CD4+ cells (median -ΔΔCt) CD8+ cells (median -ΔΔCt)IFN-γ IL-4 IL-4 TGF-b IFN-γ IL-4 IL-10 TGF-bcontrol group 10.02 -0.70 6.22 4.11 12.25 -1.17 6.69 4.15lymphoma group 8.52 -2.25 4.62 4.47 12.03 -2.39 5.72 4.70significance p=0.03 (-) (-) (-) (-) (-) (-) (-)170 W. Łuczyn´ski et al.natural killer cell leukemia/lymphoma. Leuk Lymphoma 45:2291-2294[ 4] Lee PP, Zeng D, McCaulay AE, Chen Y-F, Geiler C, UmetsuDT, Chao NJ (1997) T helper 2-dominant antilymphoma im-mune response is associated with fatal outcome. Blood 90:1611-1617[ 5] Łuczyn´ski W, Kovalchuk O, Krawczuk-Rybak M, MalinowskaI, Mitura-Lesiuk M, Chyczewski L, Matysiak M, Kowalczyk J(2005) Higher mRNA expression for TGF-beta in CD8+ cellsat the time of diagnosis of acute lymphoblastic leukemia inchildren. Pol J Environ Studies 14, Suppl 2: 283-285 [ 6] Łuczyn´ski W, Stasiak-Barmuta A, Kovalchuk O, Krawczuk-Rybak M, Malinowska I, Mitura-Lesiuk M, Olejnik I, MatysiakM, Kowalczyk J, Wieczorek M, Chyczewski L (2004) Is thereinterferon-gamma deficiency in acute lymphoblastic leukemiain children? (in Polish) Onkol Pol 7: 135-140[ 7] Łuczyn´ski W, Stasiak-Barmuta A, Krawczuk-Rybak M, Mali-nowska I (2005) Assessment of chosen costimulatory, adhe-sion, activatory molecules and cytokines of Th1/Th2 balance inacute lymphoblastic leukaemia in children. Arch Immun TherExp (in press) [ 8] Mainou-Fowler T, Proctor SJ, Taylor PR (2004) Interleukin 4production by peripheral blood lymphocytes in patients withclassical Hodgkin lymphoma. Leuk Res 28: 159-166[ 9] Mori T, Takada R, Watanabe R, Okamoto S, Ikeda Y (2001)T-helper (Th)1/Th2 imbalance in patients with previously un-treated B-cell diffuse large cell lymphoma. Cancer ImmunolImmunother 50: 566-568[10] Tsuchiya T, Ohshima K, Karube K, Yamaguchi T, Suefuji H,Hamasaki M, Kawasaki C, Suzumiya J, Tomonaga M, KikuchiM (2004) Th1, Th2, and activated T-cell marker and clinicalprognosis in peripheral T-cell lymphoma, unspecified: compari-son with AILD, ALCL, lymphoblastic lymphoma, and ATLL.Blood 103: 236-241[11] Yamasaki R, Yokota K, Okada H, Hayashi S, Mizuno M,Yoshino T, Hirai Y, Saitou D, Akagi T, Oguma K (2004)Immune response in Helicobacter pylori-induced low-gradegastric mucosa-associated lymphoid tissue (MALT) lymphoma.J Med Microbiol 53: 21-29[12] Zhang X-L, Komada Y, Chipeta J, Li Q-S, Inaba H, Azuma E,Yamamoto H, Sakurai M (2000) Intracellular cytokine profileof T cells from children with acute lymphoblastic leukemia.Cancer Immunol Immunother 49: 165-172       Received: March 18, 2005       Accepted after revision: April 25, 2005Lower levels of mRNA for IFNγ in children with lymphomas         171

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Lower expression of mRNA for interferon-gamma in T helper cells of children with newly diagnosed lymphomas

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