* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Role of Cytotoxic T Lymphocytes in Murine Cytomegalovirus Infection
Sociality and disease transmission wikipedia , lookup
Childhood immunizations in the United States wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
DNA vaccination wikipedia , lookup
Lymphopoiesis wikipedia , lookup
Molecular mimicry wikipedia , lookup
Psychoneuroimmunology wikipedia , lookup
Cancer immunotherapy wikipedia , lookup
Adaptive immune system wikipedia , lookup
Sjögren syndrome wikipedia , lookup
Schistosomiasis wikipedia , lookup
Innate immune system wikipedia , lookup
Sarcocystis wikipedia , lookup
West Nile fever wikipedia , lookup
Hospital-acquired infection wikipedia , lookup
Marburg virus disease wikipedia , lookup
Adoptive cell transfer wikipedia , lookup
Henipavirus wikipedia , lookup
Neonatal infection wikipedia , lookup
Hepatitis C wikipedia , lookup
Infection control wikipedia , lookup
J. gen. Virol. (I98O), 47, 503-508 503 Printed in Great Britain Role of Cytotoxic T Lymphocytes in Murine Cytomegalovirus Infection (Accepted 8 November I979) SUMMARY Cell-mediated immunity is important in host control of CMV infection. A chromium release microcytotoxicity assay was used to evaluate the role of cytotoxic T lymphocytes (CTL) in murine CMV infection. Within a few days after intranasal inoculation virus was detected in cultures of buffy-coat, spleens, anterior cervical lymph nodes and salivary glands. CTL were first detected on day 5 post-infection in spleen and peripheral blood, and on day 6 in anterior cervical nodes. The course of the CTL response approximated to that of virus titres during the acute phase of infection in the spleen and blood. The findings indicate that CTL are distributed to infected tissues and appear to be important during the acute, viraemic phase of infection. Cell-mediated immunity is an important part of the host defence against cytomegalovirus (CMV) infection. Human patients with suppressed cellular immunity, such as those with leukaemia, and recipients of renal, cardiac or bone marrow transplants experience frequent, often severe, infection despite their capacity to produce antibody to the virus (Betts & Hanshaw, I977). The special importance of T lymphocytes is suggested by studies of CMV infection in nude mice (Starr & Allison, 1977) and of mice treated with anti-lymphocyte serum (Jordan et al. 1977). We have previously demonstrated a cytotoxic T lymphocyte (CTL) response after intraperitoneal (i.p.) infection with murine cytomegalovirus (MCMV; Quinnan et al. T978). Recent work by Jordan provided evidence that natural infection of mice probably occurs by the upper respiratory route (Jordan, I978). In order to clarify the role of CTLs in MCMV infection we have studied their distribution in infected tissues after intranasal infection with MCMV. The Smith strain (Kim et al. I974) of MCMV was initially given to our laboratory by Dr D. Henson. Infection of mice was accomplished with an isolate that had been maintained by continuous in vivo passage, while target cells were prepared using an isolate adapted to tissue culture by ~3 in vitro passages in primary BALB/c mouse embryo cells (MEC). Weanling male BALB/c mice (Animal Production Unit, NIH, Bethesda) were inoculated intranasally under light ether anaesthesia, with 0"o5 ml of a suspension containing I x ro 4 p.f.u, of MCMV. Lymphocyte suspensions were prepared from spleens, anterior cervical lymph nodes and peripheral blood lymphocytes. Specimens taken for virus culture included anterior cervical node and spleen cell suspensions, salivary gland homogenates and samples of buffy-coats from heparinized peripheral blood. All virus cultures were performed by inoculation of specimens on to monolayer cultures of primary MEC prepared from preterm embryos. After an adsorption period of I h at 37 °C, monolayer cultures inoculated with burly coat cells were fed with minimum essential medium (MEM) supplemented with 2 % foetal bovine serum (FBS) and observed for 2 weeks for c.p.e. Other virus cultures were overlaid for plaque assay with MEM supplemented with 5 % FBS and 0"5 % agarose. After incubation at 37 °C for 72 h in a 5 % CO2 in air atmosphere, a grid was placed on the bottom of each flask and plaques were counted at 35 × magnification using an inverted Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 20:08:08 504 Short communications microscope. Target cells for cytotoxicity assays were also prepared from primary BALB/c MEC, or from passage 8 to Io cultures of MEC from the congeneric mouse strains, BIo. D2 and BIo. BR (Jackson Laboratories, Bar Harbor, Maine). The capsules of spleens and anterior cervical lymph nodes were disrupted and lymphocytes teased into suspension. Erythrocytes were removed from spleen cell suspensions by lysis with ammonium chloride buffer (Roos & Loos, I97O). Lymphocytes were separated from heparinized peripheral blood specimens by purification on Ficoll-hypaque gradients. After centrifugation, the cells were routinely resuspended in MEM with Io ~ FBS. For some studies, adherent cells were removed by passage through nylon wool columns (Julius et aL I973). In other experiments T lymphocytes were removed from cell suspension by lysis with anti-theta serum (Bionetics, Kensington, Md.) and rabbit complement (Grand Island Biological Co., Grand Island, N.Y.) as previously described (Quinnan et al. I978). The procedure used for cytotoxicity assays has been previously described (Quinnan et al. 1978). For target cells, MEC were infected at i to 4 p.f.u./cell for 24 h, harvested by trypsinization, labelled with 51Cr-sodium chromate, washed twice, suspended in MEM with I0~o FBS to a concentration of I x IOs cells/ml and dispersed in volumes ofo.I ml in wells of round-bottomed microtitre plates (Linbro, Hamden, Conn.). Equal volumes containing I × Io 6 immune lymphocytes (Ioo: I effector to target ratio) were added for test lysis, control lymphocytes for spontaneous lysis, or IO~'o Brij-35 solution for maximum lysis determinations. After incubation for I8 h at 37 °C in a 5% CO2 atmosphere, the medium was harvested using a Titertek Supernatant Collection Apparatus (Flow Laboratories, Rockville, Md.) and the released chromium measured on a gamma counter. Percentage specific immune lysis was calculated from the formula: Test c t / m i n - control ct/min × Maximum ct/min-control ct/min 1OO. Lymphocyte suspensions were prepared from tissues from two to 3° mice. Assays were performed in replicates of four to eight and results of test and spontaneous lysis were compared using the Student t-test. The results of infectivity titrations of spleen cell and lymph node cell suspensions and salivary gland homogenates are presented in Fig. I. Virus titres in spleen cell suspensions reached a maximum of 8o p.f.u./o.2 ml of cell suspension (approx. 50 × Io6 spleen cells/ml) by 6 days after initiation of infection, and no infectious virus was detected by I6 days. This period corresponded approximately to the duration of viraemia detected by culturing of buffy-coat cells, which extended from day 4 to day 2I p.i. Salivary gland virus titres did not reach maximum values until 16 days p.i. and began to decline between days 2i and 3I. Virus titres in anterior cervical lymph nodes were lower than in salivary glands, but continued to be elevated until salivary gland titres began to decline. These results thus indicate a viraemic phase lasting about 3 weeks, during which virus is also cultured from the spleen, and a chronic phase during which virus was detected locally in the salivary gland and draining lymph nodes, but not in the blood or spleen. The results of cytotoxic lymphocyte assays are also shown in Fig. I. Cytotoxic lymphocytes were first detected in spleens on days 4 to 5, produced highest immune lysis values between days 7 to I o and declined to undetectable levels by day 16. The course of the response measured in peripheral blood was similar. The CTL response in cervical lymph nodes was lower in magnitude and was usually detectable for only I or 2 days, between days 6 and 7 p.i. No significant cytotoxic lymphocyte activity was detected in cervical lymph nodes after that time. In general, the highest specific immune lysis was produced by spleen lymphocytes, but significant lysis was also produced by lymphocytes from peripheral blood and regional lymph nodes draining infected salivary glands. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 20:08:08 505 Short c o m m u n i c a t i o n s 1000000 (a) .....Q... ~. 100000 I I e? 10000 \ \\ ii I 1000 \\\ A, t / , i a00 ~ 35 - \\ iI I,~,,./ / /~'X \ \ ,, / \ / " \ 'o ,, (b) 30"~ 25 20 15- ~. 3 i I 0 l 6 9 \ z .... 12 15 18 21 24 Time p.i. (days) . 27 30 33 Fig. I. Virus infectivity titrations expressed in (a) p.f.u./o.2 ml of lymphocyte suspension or salivary gland homogenate and (b) cytotoxic lymphocyte responses during MCMV infection. (a) • - - - - • , Spleen cell suspensions; A - - - ,~, cervical node cell suspensions; [] - - [Z], salivary gland homogenates. (b) • 0 , Spleen lymphocytes; A - . - A , peripheral blood lymphocytes; D - - - F 1 , cervical node lymphocytes. Several characteristics of the cell type responsible for the immune lysis were examined and the results are presented in Table I. The cytotoxic activity of lymphocytes from all three sources was preserved in the non-adherent cell fraction obtained after passage of cells through nylon wool columns and eliminated from cell suspensions depleted of T cells by treatment with anti-theta serum and complement. Lymphocytes from all three sources were tested for their capacity to lyse MCMV-infected target cells prepared from syngeneic mice and from the congeneric mouse strains Bio. D2 and Bto. BR, which differ only in their H-2 haplotype (Klein, 1975). Effector lymphocytes produced lysis only of target cells sharing the same H-2 haplotype. Lysis of syngeneic target cells was generally greater than tysis of target cells from Bro.D2 mice, but significant lysis of both cell types was found consistently. The results presented in this study describe lymphocytes which are cytotoxic to MCMVinfected ceils and which are non-adherent, possess theta antigen and are H-2 restricted. Only T lymphocytes possess all these characteristics. These same features were typical of CTL which we have described previously in spleens of BALB/c mice infected i.p. with MCMV (Quinnan et al.Downloaded r978) andfrom which have been described in this www.microbiologyresearch.org by laboratory (Ennis et al. IP: 88.99.165.207 On: Sat, 17 Jun 2017 20:08:08 506 Short communications Table I. H-2-restricted cytotoxieity of lymphocyte suspension prepared from spleens, peripheral blood and anterior cervical lymph nodes of BALB/c mice after intranasal inoculation of M C M V Percent specific immune lysis of MCMV-infected target cells prepared from: Source of lymphocytes* Spleen Spleen Peripheral blood Peripheral blood Cervical lymph nodes Cervical lymph nodes Spleen Lymphocyte pre-treatment Nil Passage through nylon column Nil Passage through nylon column Nil Passage through nylon column Cervical lymph nodes Cervical lymph nodes Anti-theta serum and complement Complement Media Anti-theta serum and complement Complement Media Anti-theta serum and complement Complement Media Spleen Peripheral blood Cervical lymph nodes Nil Nil Nil Spleen Spleen Peripheral blood Peripheral blood Peripheral blood Cervical lymph nodes BALB/c MEC (H-2 a) BIo.D2 MEC (H-z d) B I o . B R MEC (H-2 ~) 20"9 (< 0.001)'~ 25"6 (< o.ooi) ND + ND ND ND 9'5 (< o'o5) 18"5 (< 0'04) ND ND ND ND I6"4 (< o.ooi) t4'8 (o.oo6) ND ND ND ND 8'0 (> o ' 0 ND ND ZI'O ( < 0'OOI) 26"0 (< o.ooi) o'0 (> o'I) ND ND ND ND ND ND 2I -8 ( < o.ool) 32'0 (< o'oo0 I'4 (< o'I) ND ND ND ND ND ND 8"4 (0"03o) I 1.6 (o'oo5) ND ND ND ND 20-4 (o.oo2) 29"o (< o'oor) I2.6 (o.ooz) Ii.8 (< o.ooi) 2o'3 (o'o49) 9"9 (< o.ool) o-o ( > o.I) 5'r (> oq) o'o (> o.I) * Lymphocyte suspensions used for passage through nylon yarn columns, treatment with anti-theta serum and demonstration of H-2 restriction, respectively, were obtained from spleens at I I, 8 and 8 days, from peripheral blood at 11, 7 and 6 days and from anterior cervical lymph nodes 7, 7 and 6 days p.i. t Probability by Student t-test comparing ct/min of test lysis to control lysis. ~: ND = not done. I977, I978) and by others (Doherty & Zinkernagel, I975) in a variety of murine virus infections. These results are important since they demonstrate that a CTL response occurs after infection with a herpesvirus administered by the natural, upper respiratory route, and that these cells circulate in peripheral blood and are present in regional lymph nodes draining the acutely infected salivary glands. The findings confirm the presence of CTL in critical sites during the natural course of infection and are similar to those obtained during murine influenza infection (Ennis et al. I978). The results we reported previously (Quinnan et al. I978) demonstrated that the amount of virus inoculated was proportional to the amount which replicated in the spleen and to the magnitude of the CTL response. The data included in this report indicate, however, that far fewer infectious foci are present in the spleen after intranasal inoculation than when the virus is given intraperitoneally, but that for a given dose the magnitude of the splenic CTL response is equivalent after infection by either route. In addition, the fraction of lymphocytes which are cytotoxic is similar in the spleen and the peripheral blood, but the total number oflymphocytes in the spleen is about tenfold greater. It is suggested, therefore, that the spleen is a source of CTL which may circulate through the peripheral blood to infected tissues, and that the magnitude of the splenic CTL response is proportional to the amount of virus producing the initial infection, not the amount of local virus replication. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 20:08:08 Short communications 507 The temporal relationship of the CTL response to the course of infection is also of interest. Our findings are consistent with those of others which demortstrated that viraemia begins about 3 to 4 days after the initiation of infection (Olding et al. I975; Wise et al. ~979)- In general, the duration of viraemia depends on the size of the inoculum and on the strain of mice being studied. In our experiments, virus was recovered from buffy-coat cultures during the first 3 weeks of infection, approximately the same period in which virus was detected in spleens. CTL were measured within I to z days of the initiation of viraemia and also persisted throughout the first 3 weeks. This association suggests that CTL have an important role during the acute viraemic phase of infection, but that other immune mechanisms, possibly antibody-mediated, may be more significant during the later, chronic phase of salivary gland infection. These experiments thus provide some basis for understanding one aspect of the cellular immune system, CTL, in MCMV infection. An antiviral CTL response occurs during the natural course of infection, is proportional to the virus inoculum, can be measured in peripheral blood and in proximity to infected tissues and may be important in controlling the acute, viraemic phase of infection. Since CTL cannot be detected after the acute phase of infection, studies are needed to determine the role of other cellular mechanisms, such as antibody-dependent cell-mediated cytotoxicity, in controlling chronic infection and in protective immunity. An important role for CTL in human CMV infection can only be speculative at present, but seems likely because of the many similarities between human and murine CMV infection and because of the considerable clinical evidence indicating that cellular immunity is of major importance in human CMV infection. Department of Health, Education and Welfare Food and Drug Administration Bureau of Biologics, Division of Virology 88oo RockviUe Pike Bethesda, Md. 2o2o5, U.S.A. G. V. QUrNNAN J. E. MANISCHEWITZ F. A. ENNIS REFERENCES BETTS, R. r. & HANSHAW,J. B. (Jt977). Cytomegalovirus (CMV) in the compromised host (s). Annual Review of Medicine 28, Io3-t IO. OOHERTY, t'. C. & ZINKERNAGEL,R. M. 0975). H-2 compatibility is required for T-cell-mediated tysis of target cells infected with lymphocytic cboriomeningitis virus. Journal of Experimental Medicine I4x, 5o2-5o7. ENNIS, F. A., MARTIN, W . J . , VERBONITZ, M . W . & BUTCHKO, G . M . 0977). Specificity studies on cytotoxic thymus-derived lymphocytes reactive with influenza virus-infected cells: evidence for dual recognition of H-2 and viral hemagglutinin antigens. Proceedings of the National Academy of Sciences of the United States of America 74, 3o06-3o I o. ENNIS, F. A., VERBONITZ, M. W., BUTCHKO, G. M. & ALBRECHT, P. (I978). Evidence that cytotoxic T-cells are part of the host response to influenza pneumonia. Journal of Experimental Medicine x48, t24t-I25 o. JORDAN, M. C. 0978). Interstitial pneumonia and subclinical infection after intranasal inoculation of routine cytomegalovirus. Infection and Immunity zx, 275-280. JORDArq, M. C., SHANLEY,J. D. & STEVENS,J. G. 0977). Immunosuppression reactivates and disseminates latent murine cytomegalovirus. Journal of General Virology 37, 419-423. JULIUS, M., STMPSON,E. e~ HERZENBEIG, L. A. 0973). A rapid method for the isolation of functional thymusderived lymphocytes. European Journal oflmmunology 3, 645-648. KIM, K. S., SAPIENZA,V. & CARP, R. I. (1974). Comparative studies of the Smith and Raynaud strains of murine cytomegalovirus. Infection and Immunity xo, 672-674. KLEIN, J. 0975). In Biology of the Mouse Histocompatibility- 2 Complex. New York: Springer Verlag. OLDINO, L. B., JENSEN, r. C. & OLDSTONr, ~a. B. A. (I975). Pathogenesis of cytomegalovirus infection. I. Activation of virus from bone marrow-derived lymphocytes by in vitro allogenic reaction. Journal of Experimental Medicine x4x, 56t-57z. QUINNAN, G . V . , MANISCHEWITZ, J . E . & ENNIS, E.A. 0 9 7 8 ) . Cytotoxic T lymphocyte r e s p o n s e t o r o u t i n e cytomegalovirus infection. Nature, London 273, 541-543. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 20:08:08 508 Short communications ROOS, D. & LOOS, J. A. 0970). Changes in the carbohydrate metabolism of mitogenically stimulated human peripheral lymphocytes. Bioehimica et Biophysica Acta 222, 565-582. STARR, S.E. & ALUSON, A. ¢. 0977). Role of T lymphocytes in recovery from murine cytomegalovirus infection. Infection and hnmunity I7, 458-462. WISE, T, G., MANISCHEWITZ, J. ~., QU1NNAN, G. ¥., AULAKH, G. S. & ENNIS, F. A. (1979). Latent cytomegalovirus infection of BALB/c mouse spleens detected by an explant culture technique. Journal of General Virology 44, 551-556. (Received 2o July I979) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 20:08:08