The Significance of T Lymphocytes in Transfusion Medicine

The Significance of T Lymphocytes in Transfusion Medicine Harry E. Prince

HYMUS-DERIVED lymphoeytes, or T eelIs, represent approximately 75% of the circulating lymphoeyte pool in humans. A phenomenal bank of information generated over the last 30 years demonstrates that T lymphoeytes are eentral to a properly functioning immune system. They regulate antibody produetion by B lymphocytes, seerete faetors that enhanee maerophage clearanee of eertain pathogens (eell-mediated immunity), and kill virus-infected eells. As our knowledge of T-celI funetion in immune responses has inereased, our knowledge of the impaet of T lymphoeytes on the field of transfusion medicine also has increased. This impaet ean be negative, as exemplified by transfusion-mediated transmission of viruses that infect T lymphoeytes and transfusion-assoeiated graft-versus-host disease. Another impaet coneerns the effeets of transfusion praetices on the immunoeompetence of both donors and reeipients. What is the effeet of repeated eytapheresis on donor T cells? What effect does transfusion have on reeipient T eelIs? From this latter eoncern has eome one of the positive impaets of T eells on transfusion medicine, namely, the beneficial effect of prior transfusion on renal allograft survival. Another positive impact is the emerging field of autolymphocyte therapy in whieh immunocompetent T eells are expanded ex vivo and returned to the donor patient via infusion. The purpose of this review is to summarlze for transfusion seientists the negative and positive impaets of T lymphocytes in areas relevant to transfusion medicine.

T

From the American Red Cross Blood Services, Los Angelesl Orange Counties Region, Los Angeles, CA. H.E.P. is the recipient oj an Established lnvestigator Award from the Biomedical Research and Development Program oj the American Red Cross Blood Services National Headquarters, Rockville, MD. Address reprint requests to Harry E. Prince, PhD, American Red Cross Blood Services, Los AngeleslOrange Counties Region, 1130 S Vermont Ave, Los Angeles, CA 90006. Copyright © 1992 by W.B. Saunders Company 0887-7963/92/0601-0004$3.0010

32

T LYMPHOCYTES AS THE RESERVOIR OF RETROVIRUSES TRANSMITTED BY TRANSFUSlON

Human Immunodeficiency Virus-]

The 1980s saw the emergenee of a deadly retrovirus that gradualIy destroys the immunocompetence of the hosL The clinical endpoint of this infection, termed acquired immunodeficiency syndrome (AIDS), is characterized by the development of opportunistic infections like Pneumocystis carinii and Mycobacterium avium. 1 AIDS was initially recognized in sexually active homosexual men, but was quickly doeumented in individuals whose only risk factor was receipt of a blood transfusion, which suggested transfusion-mediated transmission of the etiologie agent. 2 This hypothesis was subsequently confirmed when human immunodeficieney virus type 1 (HIV-l) was identified as the etiologie agent for AIDS 3 ; essentially all transfusion-associated AIDS eases were positive for HIV-1. 4 The ineidence of transfusion-associated AIDS has been greatly redueed by the implementation of sereening tests for antibodies against HIV-1 in blood donors. However, transmission of HIV-I ean still occur in rare situations in which the donor has been infected reeently, but has not yet produeed deteetable levels of antibodies to HIV-l. 5 The development of tests to identify donors in this "window" period is a high priority in transfusion medicine. The primary cellular target for HIV-1 infection is the CD4 T lymphocyte, 6 which serves a helper/ indueer role in regulating immune responses. CD4 T eells secrete lymphokines needed by B eelIs for optimal antibody production (ie, they help B eells), and they secrete lymphokines that induee suppressor celI function. 7 CD4 T eells are also the primary eells involved in cell-mediated immunity against a variety of pathogens (eg, Mycobacterium organisms). Eventually HIV-I kills the CD4 cells it infects, impairing the host's ability to fight infection. The number of eireulating CD4 T eells infeeted

Transfusion Medicine Reviews, Vol VI, No 1 (January), 1992: pp 32-43

33

TCELLS IN TRANSFUSlON MEDICINE

by. HIV-1 can vary tremendously; however, in 1110st asymptomatie HIV-l-positive individuals, usually less than 1 in 10,000 CD4 T cells carry viral genorne. 8 Despite this low frequency, HIV-l is transmitted with 90% efficiency via transfusion. 9 This efficiency decreases to 50% if packed red cells are stored at refrigerator temperatures for rtlore than 3 weeks suggesting that a lower number of viable infected lymphocytes is associated with less efficient transmission. 10 The primary receptor for HIV-lon the CD4 T lymphocyte is the CD4 molecule itself. This molecule specifieally recognizes the gp120 envelope glycoprotein of HIV_1. 6 Although the mechanisms of CD4 T-cell infection by HIV-1 is complex, it is elear that activation of the CD4 cell plays an important role in the infectious process. CD4 cells that have been recent1y activated, either by antigen or mitogen, are much more susceptible to HIV-l infection than resting CD4 cells. l l Because some activated CD4 cells develop into memory cells that facilitate anamnestic responses,7 it was hypothesized that the main reservoir of HIV-1 in vivo is the memory CD4-cell subset rather than the naive, or virgin, CD4-cell subset. With the advent of monoclonal antibodies to differentiate memory from naive CD4 T cells, this hypothesis was recent1y confirmed. 12 In addition to increasing susceptibility 10 HIV-I infection, activation also enhances viral replication in CD4 T cells already infected by HIV-1. 13 Two groups of investigators have shown that production of viral antigens and infectious virus by HIV-1infected CD4 cells is markedly higher following activation by antigen or mitogen. 6,14 The mechanism responsible for this enhancement apparently involves the production of transcription factors by activated, but not resting, CD4 cells. These factors normally promote transcriptionof genes coding for proteins needed for the activation cascade to proceed, such as the growth factor interleukin-2 (IL2). However, these same transcription factors also promote the transcription of HIV-I.genes, leading 10 enhanced viral replication. 15 The enhancing effects of T-cell activation on HIV-1 infectivity and replication help explain why transfusion-mediated HIV-1 transmission is highly efficient. The reciprocal allogeneic stimulation, which occurs when HIV-l-infected cells of donor origin are introduced into the recipient, results in

activation of both the infected CD4 cell and the targeted CD4 cell. These conditions allow for optimal HIV-l transmission. 16

Human T-Ce/l Lymphotropic Virus CD4 T lymphocytes can also harbor and transmit human T-cell lymphotropic virus (HTLV), a retrovirus distantly related to HIV-I. 17 Two types of HTLV have been identified; HTLV-I has been linked to adult T-cellleukemia and to a neurologieal disorder now referred to as HTLV-assoeiated myelopathy (HAM) or tropical spastic paraparesis (TSP).17 However, the vast majority (>90%) of HTLV-I-infected individuals remain asymptomatic. HTLV-II is elosely related to HTLV-I exhibiting 65% nucleotide sequence homology. 18 Disease associations with HTLV-II are less elear; onIy a few unusual cases of atypical hairy-cell leukemia have been associated with HTLV-II infection. Both HTLV-I and HTLV-II can be transmitted by blood transfusion. 19 In Los Angeles, the majority (>75%) of HTLV-positive donors are infected with HTLV-II. The major risk factor among these donors is intravenous drug use or sexual eontaet with a drug user. This finding supports that of Lee et al, who found HTLV-II infection endemic among drug users in New Orleans, LA. 2o Workers in Japan, where HTLV-I is endemie, have shown that HTLV-I is transmitted at 63% efficiency by cellular blood products 21 ; data for the effieiency of transfer of HTLV-II are not yet available. Unlike HIV-l, HTLV is not transmitted by plasma products,21 indicating a much higher level of eell association for HTLV . HTLV also differs from HIV-1 in that infeetivity of HTLV-positive products markedly decreases with storage. Donegan et al 19 reported that transmission of HTLV is almost always limited to recipients receiving platelets or paeked red cells stored for less than 8 days; packed red cells stored for greater periods of time were apparent1y noninfectious because of the death of the infected lymphoeytes at refrigerator temperatures. This assumption is supported by the experimental data af Marishima et al,22 who found that all of 20 HTLVI-positive units stored less than 7 days were still culture-positive for virus, whereas only 1 af 10 units stored for 20 days was culture-positive. Cell eulture and polymerase ehain reactian studies show that HTLV-I infects CD4 T cells but not

34

CD8 T cells (suppressor/cytotoxic), B cells, or natural killer cells. 23 Like HIV, HTLV-I infection seems to be almost totally restricted to the memory CD4 T-cell subset, suggesting that T-cell activation may similarly enhance virus infectivity. The malignant cell characterizing adult T-cellleukemia is also a memory CD4 T cell based on surface phenotype,24 which supports the findings in asymptomatic carriers and HAM/TSP patients as previously cited. Gessain et al 25 have estimated by Southern blot analysis that as many as 2% of lymphocytes (roughly 5% of CD4 cells) from asymptomatic HTLV-I carriers may be infected. This value is much higher than that observed in HIV-1 infection. 8 The cellular reservoir for HTLV-II has not been elearly delineated. Because both CD4 T-cell and CD8 T-cell lines expressing the virus have been derived from HTLV-II-positive patients with atypical hairy-cellleukemia, both lineages may be infected. 17 However, further studies are needed to determine which cells carry HTLV-II genome in asymptomatic HTLV-II carriers. T lYMPHOCYTES CAN MEDIATE TRANSFUSIONASSOCIATED GRAFT-VERSUS-HOST DISEASE

In the vast majority of transfusions, differences in the HLA antigens on donor and recipient cells leads to reciprocal allogeneic activation of donor and recipient T lymphocytes. Thus, the donorderived T cells attempt to destroy the recipient (host) cells, while the recipient T cells attempt to destroy the donor cells. Because the donor T cells are vast1y outnumbered by the recipient T cells, elimination of the donor's cells (ie, host-v-graft reaction) is easily achieved in immunocompetent recipients. 26 However, in immunocompromised hosts such as bone marrow transplant recipients, the host T cells are not able to recognize donorderived T cells as foreign. In these situations, the donor's T cells survive and systematically destroy the host's cells (graft-v-host disease [GVHD)). Immunocompromised individuals should routinely receive blood products that have been treated with gamma irradiation to inhibit the proliferative capabilities of donor T lymphocytes.27 A renewed interest in transfusion-associated GVHD (TA-GVHD) was recently precipitated by the description of TA-GVHD in immunocompetent recipients. 28 These cases apparent1y reflect the transfusion of fresh whole blood from a donor who

HARRY E. PRINCE

is homozygous for one ofthe recipient's HLA haplotypes. Thus, the recipient's T cells do not recognize the donor's cells as foreign and do not attempt to destroy them. In contrast, the donor's T cells recognize the recipient's other haplotype as foreign and are activated to destroy the recipient's cells. In some of the newly reported TA-GVHD cases, the donor was a child of the recipient, thus increased use of directed donations among family members may lead to the more frequent occurrence of this type of GVHD. The exact number of donor T cells needed to mediate TA-GVHD remains unelear. Experimental data from animal models suggest that 107 lymphocytes per kg of the recipient's body weight are required to cause GVHD. 28 However, for obvious ethical reasons, such data are not available for humans. If we work from the assumption that this figure is generally correct, then about 7 X 108 lymphocytes would be required to initiate GVHD in a 150-pound (68 kg) individual. This number of lymphocytes would be found in one to two units of whole blood or packed red cells, pooled platelet concentrate, granulocytes, or a single plateletapheresis product. Likewise, the type(s) of T lymphocytes necessary to mediate TA-GVHD in humans is not elear. Based on murine models 29 and in vitro studies of alloantigen-induced cytolytic T-cell generation,30 it seems that both CD4 (T-helper/inducer) and CD8 (T-suppressor/cytotoxic) cells are required. CD4 T cells initially recognize the HLA disparity and become activated, in tum secreting lymphokines that induce cytotoxic activity in the CD8 T-cell subset. A variety of methods are available that may be useful in preventing TA-GVHD. The most common method is gamma irradiation (500 to 2,000 rad) of the donor-derived product prior to transfusion of the recipient. As previously mentioned, gamma irradiation inhibits lymphocyte mitotic activity and blast transformation, thus donor T lymphocytes activated by the alloantigens of the recipient are not able to proliferate or mature into competent effector cells. 27 However, the functions of the other transfused cellular components are not affected by gamma irradiation. Allogeneic T-cell activation also can be inhibited by ultravi01et irradiation, which is generated without the use of radioactive isotopes. This procedure offers the advantages of increased safety for laboratory workers

T CELLS IN TRANSFUSlON MEDICINE

and wider availability. Recent studies have shown that ultraviolet irradiation of blood products prevents GVHD in a canine modee 1 ; however, its efficacy in humans remains to be determined. Another approach to preventing TA-GVHD is selective removal of lymphocytes from donorderived products prior to transfusion. Such techniques should also prove valuable in preventing the transmission of Iymphocyte-associated viruses like HIV-I and HTLV. A simple technique to reduce the number of lymphocytes in red cell products is elimination of the buffy coat celIs after centrifugation of whole blood. However, most of the efforts in lymphocyte removal have focused on filtration procedures. 28 Commercially available leukocyte filters composed of celIulose-acetate or polyester retain 95% to 99.9% of white celIs, including T lymphocytes. 32 This level of removal should be sufficient to prevent TA-GVHD. However, it remains unclear if filtration can completely prevent transmission of retroviruses. HIV-I-infected celIs, although greatly reduced in number, can still be detected in filtered units of blood from HIVinfected individuals,32 suggesting that the potential for infectivity still exists. CHAN GES IN T-LYMPHOCYTE NUMBER AND FUNCTION IN CYTAPHERESIS DONORS

The development of cytapheresis procedures allows blood centers to provide large numbers of specific celI types for transfusion. The most common procedure is plateletapheresis to provide single-donor platelets for transfusion, which reduces the level of recipient exposure to alloantigens and infectious agents. Because such procedures remove large numbers of lymphocytes from the donor, the effects of cytapheresis on the donor's immunocompetence must be a major concern of transfusion medicine. Several studies addressing this issue appeared in the early and rniddle 1980s, a time period when cytapheresis procedures removed up to 4 x 109 lymphocytes per procedure.ln 1981, Senhauser et 3 ae documented that the absolute lymphocyte Count and T lymphocyte count were 25% lower in cytapheresis donors (>6 procedures per year) than in whole blood donors. These findings were confirmed and extended in 1983 by Heal et al,34 who found that the absolute lymphocyte count was decreased 35% in cytapheresis donors and reflected decreased levels of both CD4 and CD8 T lympho-

35

cytes. A somewhat disturbing finding in both of these studies was the failure of the absolute lymphocyte count to return to normal levels after an 8-month abstention from cytapheresis procedures. This finding raised basic questions about replenishment of lymphocytes in these donors. Alater study by Matsui et al 35 similarly found reduced levels of CD4 and CD8 celIs in plateletapheresis donors, but also found a significantly decreased CD4:CD8 ratio caused by reduced absolute numbers of CD4 cells. This reduced ratio may reflect selective removal of CD4 versus CD8 celIs by the procedure, with a sirnilar replenishment rate of these two major T-Iymphocyte subsets. Alternatively, CD4 celIs may be replenished more slowly than CD8 cells. The finding that the mean CD4:CD8 ratio of lymphocytes in the plateletapheresis residues was identical to that in the peripheral blood before the procedure was started argues against selective removal of CD4 cells by the procedure thus supporting the hypothesis of differential replenishment rates for CD4 and CD8 T cells. Matsui et al 35 also assessed T-Iymphocyte proliferative responses to mitogens and alloantigens in plateletapheresis donors. Somewhat surprisingly, the phytohemagglutinin (PHA) response was higher than control donor responses, whereas the allogeneic response was lower. The mechanisms responsible for this dichotomy of proliferative responses remain unclear. Beginning in the late 1980s, the use of improved cytapheresis instruments reduced the number of lymphocytes removed from the donor to less than 1 x 109 per procedure, a value fourfold less than the number removed when the previously described studies were performed. Thus it is possible that complete lymphocyte replenishment can be achieved between procedures in these donors. However, no studies assessing circulating lymphocyte levels in donors cytapheresed using these new instruments have been published. Therefore, tbis investigator's laboratory has begun such studies. The preliminary findings are presented in Table 1. No significant differences in the absolute number of circulating lymphocytes or subsets of lymphocytes were identified suggesting that lymphocyte replenishment can occur between procedures (2 mo) ifless than 1 X 109 lymphocytes are removed per procedure. However, the findings did show a smalI but statisticalIy significant increase in the

HARRY E. PRINCE

36 Table 1. T-Lymphocyte Subsets in Cytapheresis Donors Lymphocyte Subset Ali Iymphoeytes Teells C04 eells C08 eells C016/56 eells (Natural killers) C019 eells (B eells) Proportion (%) of indieated subset expressing C045RA: C04 eells

Multiple-Product

First-Time

Donors

Donors

1659 ±587 1187 ±453 863 ±315 403 ±180 222 ± 61 168 ± 94

1797 ±533 1297 ±451 810 ±276 522 ±236 211 ± 97 180 ±138

58.9* 5.6 81.6 ± 9.2

51.6 8.1 84.2 ± 6.5

±

C08 eells

±

* Signifieantly different trom the first-time donor group (P < 0.05 student's t-test). Note: Multiple-produet donors had donated nine or more produets during the previous 24 months. Blood specimens were obtained from persons in both study groups (n = 12 per group) before that day's proeedure was begun. Lymphoeyte subsets were assessed by dual eolor-flow eytometry (FACSean). Results represent the mean celi number/""L whole blood ± 1 standard deviation.

proportion of CD4 cells expressing a marker of naive cells (CD45RA) in the cytapheresis group (Table l). As previously mentioned, naive lymphocytes represent celIs that have not yet been exposed to the antigen they are programmed to recognize. 7 It can be predicted that cytapheresis donors who undergo repeated lymphocyte replenishment will have higher proportions of naive T cells. These findings seem to confirm, at least for the CD4 T-cell compartment of circulating lymphocytes, this hypothesis. T-LYMPHOCYTE ALTERATIONS IN RECIPIENTS OF PLASMA DERIVATIVES

Soon after the discovery that AIDS was transmitted by transfusion, reports appeared describing AIDS in hemophilia patients receiving clotting factor concentrates (Factors VIII or IX) prepared from cryoprecipitates of donated plasma. 36 Because HIV-l had not been identified as the causative agent for AIDS, T-lymphocyte subsets and prolif-

erative function were monitored in hemophilia patients as indicators of immunologie changes consistent with AIDS. When serum sampies from these patients were tested retrospectively for antibodies to HIV-l, it was found that a sizable group of hemophilia patients (usually 25% to 40%) were seronegative for HIV-l. Researchers took advantage of these newly available data to assess the effects of concentrate usage in the absence of HIV-l infection on T-celI number and function in hemophilia patients. The main results of several studies are summarized in Table 2. 37-46 They generally show that levels of CD4 cells are decreased and levels of CD8 cells are increased in concentrate recipients. These changes were generally accompanied by decreased lymphocyte proliferative responses to the mitogens PHA, concanavalin A (ConA), and pokeweed mitogen (PWM); however, proliferation in response to allogeneie cells in a mixed lymphocyte reaction (MLR) were normal. A few of these studies presented data on in vivo skin test reactivity to recall antigens, and alI found marked anergy (lack of response) in HIV-seronegative concentrate recipients. Taken together, these findings were interpreted as evidence that clotting factor concentrates suppressed the immunocompetence of recipients,

Table 2. T-Lymphocyte Alterations in HIV-Seronegative Recipients of Plasma Derivatives

coa

Reference No.

CD4 Cells

Cells

37 38 39 40

Oeereased Oeereased Oeereased No ehange

Inereased Inereased Inereased Inereased

41 42

NOP No ehange

NOP No ehange

43

Oeereased

No ehange

44

Oeereased

Inereased

45 46

No ehange NOP

Inereased NOP

Proliferative Responses NOP NOP NOP Oeereased PWM, normai PHA ConA Oeereased PWM Normai PHA, ConA, MLR Normai PWM, PHA, ConA, deereased skin test Oeereased PWM, PHA, ConA normai MLR NOP Oeereased ski n test

Abbreviations: NOP, no data presented. Note: C04 and CD8 eolumns refleet changes in absolute number or Iymphoeyte pereentage expressed as either signifieant differenees in mean values versus eontrols or proportion of patients with abnormal results.

37

T CELLS IN TRANSFUSlON MEDICINE

perhaps rendering them more susceptible to HIV-I or other infectious agents. It remains unclear if the same mechanisms are responsible for altered T-cell subsets and reduced T cell proliferative potential in concentrate recipients. The increased levels of CD8 cells observed most likely reflected elevated levels of CD8 cell subsets that express markers indicative of activation, such as HLA-DR. 44 It is hypothesized that these activated CD8 cells are generated in response to chronie antigenic stimulation by proteins found in the eoncentrate preparations. Sueh proteins include the clotting factor itself and fibronectin, fibrinogen, HLA proteins, and any viral antigens that are undoubtedly present in pools made from sueh a large number of individuals. In contrast, the decreased proliferative activity of T cells from concentrate recipients is hypothesized to reflect the action of an inhibitory component in concentrates. In vitro data supporting this hypothesis were published by Froebel et al,47 who found that both Scottish and American factor VIII concentrates inhibited T-cell proliferation induced by PHA or ConA. Lederman et al 48 extended these findings by showing that factor VIII preparations inhibit the in vitro proliferative response of T lymphocytes induced by the soluble antigen tetanus toxoid as well as by PHA. They further found that an early event in the T-cell activation cascade was inhibited by eoncentrate; cells exposed to concentrate for only the first 4 hours of culture showed decreased proliferative responses. Similarly, Schreiber et al49 showed that factor VIII concentrates inhibited allogeneic proliferative responses of T lymphocytes (MLR). Because clotting factor concentrates consist almost entirely of extraneous plasma proteins (99%) rather than the clotting factor of interest « 1%), it was suspected that the component inhibiting T lymphocyte proliferative responses is one or more of these extraneous proteins and not the clotting factor. Data supporting this hypothesis were obtained by Schreiber et al,49 who found that factor VIII purified by the use of a specific monoclonal antibody did not inhibit T cell proliferative responses to mitogen or alloantigen at any concentration tested. This product is now being tested to determine if recipients of this purified factor VIII exhibit the changes in T-cell subsets and proliferative function observed in recipients of conventional concentrate. 50 Similar studies are also being conducted with recombinant factor VIII. 51 Such studies should help define the mech-

anisms responsible for changes in T-cell subsets and function in concentrate recipients. Ii factor VIII is not the concentrate component inhibiting T-cell proliferation, what is? A series of well-conducted experiments by Schultz and Shahidi 52 ,53 indicate that fibronectin may be the major inhibitory protein in plasma and clotting factor cryoprecipitates prepared from plasma. These investigators showed that plasma depleted of fibronectin by either affinity chromatography or cryoprecipitate preparation was much less effective at inhibiting PHA-stimulated T-cell proliferation when compared with untreated plasma. Further, the inhibitory activity was recovered in resolubilized cryoprecipitate, a mixture consisting of heparin, fibrinogen, and fibronectin. When these components were tested in purified form for inhibition of T cell responses to PHA, only fibronectin was inhibitory. Purified fibronectin also effectively inhibited allogeneic T-cell activation in an MLR. Further studies explored the mechanism of fibronectin inhibition of T-lymphocyte activation. The results showed that fibronectin, like concentrate, 48 inhibited an early event in the activation cascade and did not act by inducing the release of a suppressive factor from accessory cells (monocytes). Further, fibronectin did not interfere with IL-2 use by aetivated cells; however, IL-2 production was not measured. Studies have begun at this institution to further characterize the mechanism of plasma-mediated inhibition of T-cell activation. We have found that plasma inhibition of PHA-induced DNA synthesis is paralleled by inhibition of lymphoeyte IL-2 receptor (CD25) expression (Fig lA). Further, plasma inhibited PHA-induced CD25 expression by both of the major T-Iymphocyte subsets, CD4 and CD8 lymphoeytes (Fig IB). Beeause CD25 expression is an early event in the T-cell aetivation eascade, these results are in eoncordance with other studies 48 ,52,53 showing that plasma exerts its inhibitory effect early in the activation proeess. Additional studies will determine if plasma inhibits CD25 expression directly or indireetly by inhibiting an even earlier event that is prerequisite for CD25 expression. T-LYMPHOCYTE ALTERATIONS IN RECIPIENTS OF CELLULAR BLOOD PRODUCTS

Studies to determine the effects of the cellular eomponents of transfused blood on the recipient's

HARRY E. PRINCE

38

100

10 100 e CD25 + LYMPHOCYTES o DNA SYNTHESIS

fi> 90 W ~

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80

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~ ~

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70

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IJL PLASMAlCULTURE

o

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20

30

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lJl PLASMA/CULTURE

Fig 1. Inhibitory effects of autologous plasma on PHA-induced T·lymphocyte activation. Peripheral blood mononuclear cells were cultured In microtiter wells with PHA in the presence of various amounts of autologous plasma (O to 50 !Jol plasma plus RPMI culture medium containing 5% human AB serum to atotal volume of 200 !Jol per well). Wells for measuring DNA synthesls were pulsed with 3H-thymidine for 4 hours before harvestlng on day 3 of culture. Cells from additional wells were harvested on day 3 and stained wlth phycoerythrin-tagged anti-CD25 and either fluorescein-tagged anti-CD4 or antl-CD8. then analyzed using a FACScan flow cytometer. (Al Plasma inhibition of PHA-induced DNA synthesis (cpml is paralleled by inhibitlon ot CD25 expression by Iymphocytes. (B) Plasma inhibits PHA-induced CD25 expression by both CD4 and CD8 cells. For all parameters. values in the absence ot PHA were the same as those observed in the presence of PHA plus 50 !JoL plasma,

immune system have been conducted in three general patient groups. One group consisted of patients transfused during surgery. However, as pointed out in an excellent review by Triulzi et al,54 these studies are complicated by the immunologie consequences associated with surgical trauma and anesthesia. A second study group consisted of patients with hematologic diseases such as sickle cell anemia and thalassemia. These studies likewise are complicated by observations showing altered T-cell subsets and proliferative potential in nontransfused patients with these diseases. For example, Kaplan et al 55 found that the absolute numbers ofboth CD4 and CD8 T-cell subsets were elevated in transfused sickle cell anemia patients; however, absolute CD8 cell levels were also increased in nontransfused sickle cell patients. As a group, transfused thalassemia patients do not exhibit alterations in absolute CD4 and CD8 T-cell levels; however, proliferative responses to mitogens and soluble antigens are apparently decreased in this group. 56,57 The largest patient group used to study the effects of transfusion on the immune response con-

sists of patients with renal disease. The reason for this is the discovery in the 1970s that b100d transfusion enhanced renal allograft survival, which is referred to as the transfusion effect. 58 It was found that allograft recipients who received transfusions prior to transplantation were much less likely to reject their allografts as compared with nontransfused recipients. This phenomenon was attributed to transfusion-induced immunologic unresponsiveness of the recipient; thus, a large body of work has been performed as part of attempts to define the mechanisms responsible. Studies to assess transfusion-associated changes in circulating T-cell subsets in patients with renal disease have yielded conflicting data. For example, one study found reduced levels of both CD4 and CD8 cells and no subsequent change in CD4:CD8 ratio in al1 patients undergoing hemodialysis regard1ess of their past transfusion history.59 In contrast, another study found a se1ective decrease in CD4 cell numher in transfused versus nontransfused hemodialysis patients. 6O The source of this inconsistency remains unclear. In the absence of profound numerical changes in

T cELLS IN TRANSFUSlON MEDICINE

T-cell subsets following transfusion, investigators have focused on the identification of functionał changes that might explain the transfusion effect. One theory states that transfusion simply serves to immunize the patient awaiting transplantation, and that the immunosuppressive drugs given at the time of transplantation eliminate donor-specific 61 clones of responding T cells. Terasaki based this clonał deletion theory on two basic characteristics of the transfusion effect: (l) the blood used for transfusion must share HLA antigens with the donated organ (in fact, blood from the potentiał organ donor is the most effective); and (2) the transfusion effect is seen only in combination with immunosuppressive drug treatment (azathioprine and prednisone were the drugs in use at the time the theory was proposed). The theory argues that transfusion primes the recipient to HLA antigens ofthe donor, generating donor-specific memory T cells. Following organ transplantation, these memory T cells are swiftly activated as part ofthe host's attempt to eliminate the graft. These activated celIs are acutely sensitive to the immunosuppressive drugs and are destroyed as they attempt to do their job. This theory suggests that no real functional alterations in T cells are induced by transfusion; rather, any changes reflect the normal response to foreign antigens. Data supporting Terasaki's clonal deletion theory were presented by Vandekerckhove et al. 62 These investigators found an increase in the number of donor-specific T-helper celI precursors (CD4) and cytotoxic T-cell precursors (CD8) following donor-specific transfusion given to men awaiting kidney transplantation. Thus, it seems that transfusion does indeed immunize the host against donor antigens. However, there are problems with the elonal deletion theory as the sole explanation for the transfusion effect. First, there is elear evidence that organ-specific immunoreactive elones are not alI deleted. Biopsies show cytotoxic celIs of host origin infiltrating long-functioning successful grafts .63 Second, spouse-specific transfusion has been shown to be an effective means of treating women with a history of recurrent spontaneous abortions; however, this treatment does not inelude immunosuppressive drugs. 64 Terasaki suggests that endogenous corticosteroids rather than immunosuppressive drugs are responsible for elonał deletion in this system. However, as asserted by Per-

39

kins, it is difficult to believe that endogenous corticoids could mediate complete elonal deletion. 63 A second and more accepted theory to explain the transfusion effect is transfusion-mediated induction of suppressor T lymphocytes. Early studies demonstrated reduced T-cell proliferation to mitogens, alloantigens, and soluble antigens in transfused patients with end-stage renał disease65 that suggested a nonspecific suppressor effect. Other studies demonstrated only donor-specific suppressor cells when tested in MLR. 66 These conflicting data were partly resolved by Smith et al, who found that nonspecific suppressor activity in transfused hemodialysis patients was a transitory phenomenon that peaked at 3 weeks posttransfusion, but was undetectable by 20 weeks posttransfusion. 67 With the discovery that donor-specific transfusions were most efficient for generating the transfusion effect,63 most studies conducted in recent years have focused on identifying donorspecific suppressor T cells and defining their mechanism of action. Investigators at Oxford University used a rat model to demonstrate conelusively the generation of donor-specific suppressor T cells in transfused animałs. 68 Further, these cells were effective at preventing graft rejection in vivo. Strain DA animałs were transfused with strain LEW blood; 7 days later, lymph node cells from these transfused DA animals were transferred to syngeneic DA nontransfused animals. When these animals .received a LEW kidney, no rejection occurred; however, if these animals received a strain PVG kidney, the organ was rejected. Because such controlled studies are not possible in humans , Loertscher and Strom69 developed an in vitro MLR system to generate donor-specific suppressor T cells and have used it to dissect the mechanisms operating. An MLR containing heattreated stimulator cells and fresh responder cells is cultured for 8 days, generating respondersuppressor cells specific for stimulator HLA antigens. These cells are then irradiated and added to a fresh MLR using cells from the same stimulator and responder. These investigators found that the suppressor cells inhibited the ability of both CD4 and CD8 responder cells to be activated by the stimulator cells. The parameters measured were IL-2-receptor and transferrin-receptor expression, events known to be upregulated by IL-2. This find-

40

ing suggested that the suppressor celIs may be exerting their immunosuppressive effect by inhibiting IL-2 production by responder CD4 cells. To test this hypothesis, Loertscher and Strom added recombinant IL-2 to the suppressor cell assay. They found that responder CD8 celIs now expressed normallevels of IL-2-receptor and transfemn-receptor, but that responder CD4 celIs did not. Hence, donor-specifie suppressor celIs seem to target responder CD4 celIs, inhibiting their activation, and thus their ability to produce IL-2; without the CD4 cell-deńved IL-2, responder CD8 celIs also are unable to respond to alloantigens. Together, these findings suggest that transfusion induces alloantigen-specifie helper and cytotoxic T cells, but also induces suppressor T celIs that inhibit the activation of these helper and cytotoxic T cells when the alloantigen is again ptesented. The mechanism of suppressor cell function is inhibition of IL-2 production by the alloreactive CD4 cells, and without this IL-2, the alloreactive cytotoxie T cells cannot function. This hypothesis suggests that exogenous IL-2 supplied at the time of transplantation would allow the alloreactive cytotoxic T cells to function normally and lead to graft rejection. Although such studies are ethicalIy precluded in humans, the Oxford group68 tested this hypothesis in their rat model. They found that transfused animals that received IL-2 following transplantation rejected their grafts in a time period similar to that observed in untransfused allograft recipients (median survival time of 10 days in both groups); in contrast, transfused animals not receiving IL-2 showed graft survival greater than 50 days.70 Thus, the beneficial effect of transfusion on renal allograft survival was reversed by IL-2 administration following transplantation. Whereas the above data strongly suggest that suppressor cell-mediated immunologie unresponsiveness is a major mechanism of the transfusion effect, they do not exclude clonal deletion mechanisms acting at a different stage. Perhaps the most reactive donor-specific clones (recognizing the most immunogenic HLA disparity?) are eliminated by immunosuppressive drug therapy early after transplantation and then suppressor cells downregulate the activation of clones recognizing less immunogenic HLA antigens. Further studies in inbred animai models are needed to determine if a combination of mechanisms may explain the transfusion effect in the outbred human system.

HARRY E. PRINCE

Which cells in the transfused blood are responsible for inducing the transfusion effect? Early studies conducted in Europe indicated that leukocytes are required because leukocyte-free red cells prepared by filtration did not mediate the transfusion effect. 71 Additional data supporting this hypothesis came from studies showing that transfusion of buffy coat cells led to the beneficial effect. 72 Because leukocytes express both HLA class I (ABC) and class II (HLA-DR) antigens, it was unclear if one or both types of HLA antigens were required for inducing the transfusion effect. Studies by Chapman et aC 3 showed that tra.'1sfusion of platelets expressing only class I antigens did not improve allograft survival; thus leukocytes expressing HLA-DR are the prime candidates to be the critical cells inducing the transfusion effect. Iwaki et a174 found that an HLA-DR mismatch is required for the beneficial effect of blood transfusion on alIograft survival. It remains unclear whieh HLA-DR + leukocytes are the most important for inducing the transfusion effect. Whereas only about 10% to 15% of peripheral blood T lymphocytes express HLA-DR, essentially all B cells, monocytes, and dendritic cells express HLA-DR. Because the dendritic cell is now considered the main stimulator cell for MLR,75 alogical hypothesis is that the dendritic celI is the most important for inducing the transfusion effect. TRANSFUSlON OF AUTOLOGOUS T LYMPHOCYTES EXPANDED BY EX VIVO CULTURE

A relatively new area of transfusion medicine is autolymphocyte therapy. The most impressive applications to date have been in the treatment of metastatic carcinomas and melanomas. In the protocol described by Osband et al,76 peńpheral blood mononuclear cells from patients were activated in vitro by a monoclonal antibody recognizing the T-celI-receptor for antigen. After 3 days, the supematants from these cultures were harvested, divided into several aliquots, and frozen. Later, more mononuclear cells were obtained from the patient and cultured for 5 days in a medium containing the previously generated autologous supernatant (25% v/v). This celI preparation, consisting largely of activated T cells, was retumed to the patient via infusion. The survival time of the autolymphocyte group was 2.5-fold higher than that

41

.T CELLS IN TRANSFUSlON MEDICINE

of the controi group. These investigators postulated that tumor-specific T cells were activated by incubation in the autologous lymphokine mixture. A slight1y different approach being undertaken by other investigators is the ex vivo expansion of lymphocytes found inside melanomas and carcinomas. 77 Previous studies have shown that the tumor infi1trating lymphocyte (TIL) population can be expanded using mitogens and lymphokines, and the expanded population contains cytotoxic T cells specific for the tumor. Although most investigators find that these expanded TIL express CD8, others find that TIL express CD4. 77 In a recent report by Rosenberg et al,78 infusion of expanded TIL followed by recombinant IL-21ed to regression of the cancer in 11 of 20 metastatic melanoma patients. Other investigators have begun to refine the TIL expansion system by isolating the effector cells before expansion. TIL from renal cell carcinomas, routinely CD8 + cytotoxic T cells, are selected using anti-CD8 monoclonal antibody-coated tissue culture flasks. These are then expanded by culturing in recombinant IL-2 and yield essentially pure cultures of CD8 + tumor-specific cytotoxic T cells. 79 Clinical studies are being conducted to assess the efficacy of these TIL expanded ex vivo for mediating tumor regression. A similar approach is also being investigated to expand and reinfuse autologous CD8 cells in an effort to slow the progression of HIV-l-related disease. Wiviott et al 80 found that CD8 T cells

from HIV-1-infected individuals can inhibit HIV-1 replication within infected CD4 cells via a noncytotoxic mechanism. These investigators hypothesize that greater numbers of CD8 cells may be more effective at slowing the rate of HIVinduced CD4 cell destruction in vivo and improve the patient's ability to fight opportunistic infections. Studies are underwal l to determine if autolymphocyte therapy with these CD8 cells, selected and expanded ex vivo by culture in recombinant IL-2, can improve the lifespan and quality of life of HIV-infected individuals. CONCLUSION

As this summary has shown, T lymphocyte biology can have a significant influence on the practice of transfusion medicine. Technological advances in lymphocyte removal, recombinant protein production, and inactivation of allogeneic responses are helping to eliminate some of the negative impacts of T lymphocytes on transfusion medicine. With these problems solved, transfusion scientists should be able to focus their attention on the positive aspects of T lymphocytes in transfusion medicine. Autologous and homologous T lymphocyte therapy will have many applications for treating immunodeficiencies and autoimmunity . Transfusion medicine will play an integral role in the development of treatment regimens for immune system disorders.

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66. Leivestad T, Thorsby E: Effects of HLA-haploidentical blood transfusions on donor-specific immune responsiveness. Transplantation 37:175-181, 1984 67. Smith MD, Williams JD, Coles GA, et al: Blood transfusions, suppressor T cells, and renal transplant survival. Transplantation 36:647-650, 1983 68. Quigley RL, Wood KJ, Morris PJ: Transfusion induces blood donor-specific suppressor cells. J Immunol 142:463-470, 1989 69. Loertscher R, Strom TB: Differential regulation of activation-associated receptor expression on CD4- and CD8positive T lymphocytes by allosensitized suppressor T cells. Transplantation 48:473-478, 1989 70. Dallman MI, Wood KJ, Morris PJ: Recombinant interleukin-2 (11.-2) can reverse the blood transfusion effect. Transpl Proc 21: 1165-1167, 1989 71. Persijn GG, Van Leeuwen A, Parlevliet J, et al: Two major factors influencing kidney graft survival in Eurotransplant: HLA-DR matching and blood transfusion(s). Transpl Proc 13:150-154, 1981 72. Okazaki H, Takahashi H, Mivra K, et al: Significant reduction of sensitization and improved allograft outcome with donor-specific buffy coat transfusion. Transplantation 37:523525, 1984 73. Chapman JR, Ting A, Fisher M, et al: Failure of platelet transfusion to improve human renal allograft survival. Transplantation 41:468-473, 1986 74. Iwaki Y, Cecka JM, Terasaki PI: The transfusion effect in cadaver kidney transplants-Yes ar no. Transplantation 49:56-59, 1990 75. Santiago-Schwarz F, Bakke AC, Woodward JG, et al: Further characterization of low density mononuclear cells: FACS-assisted analysis of human MLR stimulators. J Immunol 134:779-785, 1985 76. Osband ME, Lavin PT, Babayan RK, et al: Effect of autolymphocyte therapy on survival and quality of life in patients with metastatic renal-cell carcinoma. Lancet 335:994998, 1990 77. Topalian SL, Solomon D, Avis FP, et al: Immunotherapy of patients with advanced cancer using tumor-infiltrating lymphocytes and recombinant interleukin-2: A pilot study. J Clin Oncol 6:839-853, 1988 78. Rosenberg SA, Packard BS, Aebersold PM, et al: Use of tumor-infiltrating Iymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. N Engl J Med 319:1676-1680, 1988 79. Moody DJ, Feeney LA, Tso CI., et al: Large-scale selection and expansion of CD8 + tumor infiltrating Iymphocytes. Clinical Immunology Society Fifth Annual Meeting, November, 1990 (abstr 32) 80. Wiviott LD, Walker CM, Levy JA: CD8 + lymphocytes suppress HIV production by autologous CD4 + cells without eliminating the infected cells from culture. Cell Immunol 128:628-634, 1990 81. Armstrong J, Ho M, Herberman R, et al: A phase I study of autologous, activated CD8 + Iymphocytes expanded in vitro infused into patients with advanced ARC or AIDS. Sixth International Conference on AIDS, June, 1990 (abstr SB491)