the adverse consequences of the acquired immune deficiency syndrome ( aids ) or t cell immunodeficiency provide evidence of the vital role of cytotoxic t - lymphocytes ( ctl ) in the immune response .
indeed , ctl are well - known elements of the immune response to virus - infected , tumour and allogeneic cells [ 13 ] .
more recently , the role of ctl was expanded significantly when the ability to mediate direct killing of microbial pathogens was identified .
research defining the precise mechanisms underlying ctl killing of microbes , however , is still in its infancy .
remarkably , granulysin ( in contrast to granzymes ) has emerged as a fundamental mediator of microbial killing .
the mode of action of granulysin appears to be through the disruption of membrane permeability .
it follows that granulysin has been found to insert into the microbial membrane through ionic interactions between the positively charged amino acid residues and negatively charged phospholipids .
insertion of granulysin in turn disrupts membrane permeability resulting in the influx of fluid into the cytoplasm and death by osmotic lysis .
other mechanisms identified in the killing of tumor cells may also play a role , including ca influx and k efflux , and activation of a sphingomyelinase associated with the cell membrane to generate ceramide .  
ctl killing of extracellular pathogens involves direct microbial recognition by the ctl ( figure 1(a ) ) .
in contrast to tumour and virus - infected cells , recognition of extracellular pathogens occurs through an apparent mhc - independent mechanism ( as microbes have not been found to express mhc ) .
successful recognition induces the release of granulysin which may directly bind and kill the microbe . in the case of intracellular microbes
these interactions , like recognition of tumor and virus - infected cells , are often mhc - restricted and antigen - specific .
binding triggers the release of granulysin , which must enter the infected host cell for microbial killing to occur ( figure 1(b ) ) .
this is thought to be mediated through perforin - generated pores in the host cell membrane .
these pores facilitate the influx of extracellular ca which triggers the target cell to endocytose the damaged region of the membrane and internalize nearby granulysin [ 79 ] .
alternatively , ctl may mediate lysis of the infected host cells releasing microbes which may then be recognized and killed by nearby ctl ( figure 1(c ) ) .   to acquire microbicidal activity ,
this priming event can occur in response to cytokines such as t cell growth factors , stimulation by a mitogen , or antigen - specific responses .
the receptors and signalling pathways involved in priming may be the same , but also might be quite distinct from the receptors and signalling that lead to immediate killing . following the priming event ,
the microbicidal ctl is then ready to receive the signal that triggers them to kill the pathogen .
t cells from mice immunized with p. aeruginosa polysaccharide were stimulated with macrophages from nonimmune mice with or without the addition of heat - killed bacteria .
macrophages had to be present during the priming , but surprisingly , heat - killed bacteria did not .
this suggested that the bactericidal activity of these t cells was dependent on their interaction with macrophages but did not require presentation of p. aeruginosa antigens .
supernatants collected from immune t cells exposed to macrophages and p. aeruginosa were found to kill p. aeruginosa in addition to staphylococcus   aureus and escherichia coli suggesting that these t cells were producing a soluble bactericidal product .  
ctl have also been found to mediate killing of mycobacterium tuberculosis through the release of bactericidal products [ 1113 ] .
m. tuberculosis growth was reduced as much as 74% in the presence of cd4 ctl and 84% in the presence of cd8 ctl .
when cd8 ctl were pretreated with strontium chloride ( which depletes granule contents ) , it caused a clear reduction in the ability to kill m. tuberculosis that correlated with a marked decrease in granulysin content . in vitro ,
purified granulysin was able to kill extracellular m. tuberculosis in a dose - dependent fashion .
however , killing of intracellular m. tuberculosis required the addition of perforin , which was not directly bactericidal , but was able to lyse m. tuberculosis - infected macrophages . together
this data suggests that perforin facilitates entry of granulysin into m. tuberculosis - infected cells where granulysin can access and kill the intracellular pathogen . in vivo studies
have provided additional support for the conclusion that perforin is required for cd8-mediated clearance of m. tuberculosis . in these studies ,
irradiated mice infected with m. tuberculosis were found to have a significantly greater bacterial load after receiving adoptively transferred perforin - deficient cd8 t cells compared to wild - type cd8 t cells .
studies conducted with listeria innocua support a perforin - independent mechanism in which granulysin is actively taken up by the infected cell . both healthy and l. innocua - infected dendritic cells ( dc ) were found to take up recombinant granulysin in a temperature - sensitive manner , indicative of active internalization . furthermore , cholesterol depletion abrogated granulysin uptake and killing of infected dc , suggesting lipid raft involvement .
this was further supported by data showing colocalization of granulysin and cholera toxin ( used as a marker of lipid rafts ) during and shortly following uptake .
ninety minutes after uptake , granulysin was found to colocalize with phagosomes containing l. innocua dna .
indeed , granulysin was found to kill both extracellular l. innocua and l. innocua - infected dc in a dose - dependent manner . while granulysin may function independently ,
another study found that perforin treatment ( whether simultaneous or sequential to granulysin treatment ) augmented granulysin - dependent killing of intracellular l. innocua which was not due to the formation of stable pores in the dc membrane .
rather , perforin treatment was found to stimulate a transient change in the plasma membrane permeability ( assessed by ca influx ) that enhanced fusion of granulysin - containing endosomes with phagosomes containing l. innocua .  
the highest level of expression of granulysin occurred in patients afflicted with the localized tuberculoid form , rather than the disseminated lepromatous form of the disease .
this correlation suggests that granulysin release by ctl may limit the spread and severity of m. leprae infection .
cd4 t cell lines derived from tuberculoid leprosy lesions were found to lyse m. leprae infected macrophages and kill intracellular mycobacteria .
strontium treatment abrogated both the cytolytic and bactericidal activity of these cd4 t cells , which suggested that killing was mediated through the granule - exocytosis pathway . blocking fas using anti - fas antibody
also partially inhibited the cytolytic activity of two of four cd4 t cell lines suggesting that the fas - fasl pathway may also play a minor role in the lysis of m. leprae - infected cells .
it follows that the extent of fas - fasl mediated lysis may depend on the nature of the ctl   or the infected target cell .  
t cells stimulated with phytohemagglutinin ( pha ) , a mitogenic lectin , as well as various oxidative mitogens , antigens , and alloantigens were found to kill schistosomula .
killing was found to correlate with increased binding to schistosomula suggesting a contact - dependent mechanism . in support of this , supernatants from pha - stimulated t cells were ineffective at killing , although the concentration of parasiticidal products in the supernatants may not have been high enough to mediate an appreciable response . moreover , it remains unclear how pha induces ctl parasiticidal activity .
removal of pha prior to incubation of t cells with schistosomula was found to impair killing suggesting that pha may have an ulterior function in addition to stimulating t cell activation and proliferation .
ctl killing of entameoba histolytica closely resembled that of schistosoma   mansoni in many respects .
first , nonspecific activation of t cells with pha was found to significantly enhance killing of e. histolytica trophozoites compared to unstimulated t cells .
pha - stimulated t cells killed as many as 92% of trophozoites whereas amoebic viability in the presence of unstimulated t cells remained relatively unchanged .
third , and surprisingly , killing required the continuous presence of pha , again alluding to direct participation .
however , bridging the t cells and entameoba can not be the only mechanism by which pha participates in killing because at least 18 hours of preincubation was required to elicit killing .
similarly , in studies with s. mansoni , pretreatment of schistosomula with pha was not sufficient to induce parasiticidal activity of unstimulated t cells .
thus , pha appears to act both by activating the t cells as well as possibly functioning as a bridge for binding .
specific activation of t cells isolated from patients with an amoebic liver abscess with an e. histolytica lysate also induced killing .
in contrast to prior studies with pha [ 20 , 21 ] , stimulation with antigens from e. histolytica was found to augment binding intensity of t cells to the trophozoites rather than the binding frequency .
thus , the more important determinants in killing amoeba may be the binding characteristics of the ctl rather than the number of ctl bound .
anti - tnf- antibody blocked killing of immune t cells from mice immunized against e. histolytica , although large doses of tnf- did not directly kill the amoeba .
these results demonstrate the complexity of the system and the possible role of additional mediators .   
t cells from mice immunized with p30 ( a t. gondii membrane protein ) and subsequently restimulated in vitro with p30 were found to kill extracellular t. gondii . unlike studies conducted with s. mansoni and e. histolytica [ 20 , 21 ] , killing of t. gondii appeared to be antigen specific .
p30-antigen - specific splenocytes killed significantly more t. gondii than splenocytes stimulated with the mitogenic lectin , concanavalin a ( cona ) , despite greater t cell proliferation in response to cona .
although antigen stimulation resulting in microbicidal priming was likely to have been mhc - restricted , killing was direct . since there is no evidence for expression of mhc or mhc - like molecules by t. gondii , this suggests that direct cytotoxicity was mhc - independent .
mhc - unrestricted t cell killing of extracellular t. gondii has also been reported by others .
however , the mechanism by which antigen - specificity is accomplished in the absence of mhc is not yet defined .
ctl responses against intracellular t. gondii , on the other hand , have been found to proceed through an mhc - restricted mechanism .
splenocytes from mice immunized against t. gondii mediated killing of both infected macrophages and intracellular t. gondii [ 26 , 27 ] .
concanamycin , an inhibitor of the vacuolar atpase that is required to maintain perforin in lytic granules , significantly impaired killing , implicating perforin in the response against t. gondii .
perforin - deficient mice chronically infected with t. gondii were found to have a higher rate of mortality and a greater number of brain cysts compared to wild - type mice , although other studies came to disparate conclusions .
moreover , the ability of cd8 t cells to kill intracellular t. gondii is controversial . in one study ,
cd8 t cells failed to kill t. gondii - infected b cells as assessed by quantitative pcr of the sag-1 gene .
 t cells were found to kill blood - stage p. falciparum through a contact - dependent mechanism [ 3133 ] .
killing appeared to be stage - dependent with extracellular merozoites being the suggested targets [ 31 , 32 ] . in vitro studies revealed a correlation between the parasiticidal activity of the  t cells and expression of granulysin .
together , this data indicates that parasiticidal ctl mediate killing of p. falciparum via the release of granulysin .
several studies have reported the ability of ctl to kill the opportunistic fungus candida albicans .
il-2 stimulation of murine splenocytes was found to induce fungicidal activity against c. albicans as well as several other candida species .
anti - candida activity required at least 3 days of priming with il-2 and peaked at 7 days .
the induction of fungicidal activity correlated with enhanced killing of an nk cell - resistant tumor cell line .
supernatants taken from il-2 stimulated splenocytes had no effect on the growth of c. albicans suggesting a contact - dependent fungicidal mechanism .
moreover , another study found that the cd8 t cells , and not nk1.1 cells ( nk cells ) , mediated fungicidal activity against c. albicans in response to il-2 .  
the ctl response against the yeast - like pathogen cryptococcus neoformans has been described extensively . in one of the earlier studies ,
lymphoid cells were isolated after mice had been immunized with heat - killed c. neoformans in complete freund 's adjuvant .
the anticryptococcal activity of these cells was measured by assessing colony forming units , and delayed - type hypersensitivity was assessed by footpad swelling following administration of c. neoformans antigen . only lymphoid cells from immunized mice
furthermore , the magnitude of the anticryptococcal activity correlated with the intensity of the delayed - type hypersensitivity response .
t cells within the peripheral lymphoid compartment were found to be the mediators of the anticryptococcal activity . in another study , t cells
were observed to interact directly with c. neoformans in vitro suggesting a contact - dependent mechanism of killing ( figure 2 ) .
however , between 11 and 35% of t cells were found to bind to c. neoformans suggesting recognition by a receptor other than the t cell receptor .
other studies reported similar killing with human peripheral blood mononuclear cells ( pbmc ) stimulated with dead c. neoformans   or cultured for 7 days with il-2 and gm - csf ( but not with tnf , ifn- ,   or vitamin d3 ) . in agreement with prior studies [ 36 , 37 ] , t cells ( as well as nk cells )
isolated from the pbmc were found to directly bind to , and possess fungicidal activity against c. neoformans after priming with il-2 .
another study found that freshly isolated t cells could mediate killing of c. neoformans without the need for il-2 stimulation .
however , t cells cultured without il-2 soon lost their ability to kill , suggesting that il-2 is required for t cells to maintain their fungicidal activity .
treatment of t cells with the proteases trypsin and bromelain , which have been found to cleave several receptors on t cells , also impaired killing of c. neoformans suggesting that receptor - ligand interactions were involved in anticryptococcal activity .  
investigators have asked whether ctl kill cryptococcus or exert their anticryptococcal activity by inhibiting the growth of the organism .
studies using the viability dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) showed that c. neoformans failed to demonstrate metabolic activity after incubation with t cells . c. neoformans also becomes permeable and labels with propidium iodide after incubation with ctl providing further evidence for killing ( unpublished observations , anowara islam ) .
furthermore , ctl can reduce the burden of organisms below the starting innocula [ 41 , 44 ] , and consistent with previous studies that suggested a contact - dependent mechanism of killing , separation of t cells and c. neoformans with a porous membrane during incubation was found to abrogate anticryptococcal activity .
early studies investigating the mechanism of ctl anticryptococcal activity examined possible receptors and effector mechanisms .
antibody - mediated blockade of various t cell surface molecules such as lfa-1 and cd3 did not significantly inhibit killing .
together this data suggests that ctl killing of c. neoformans may proceed through a novel receptor that is yet to be identified .  
these early studies also examined the role of reactive hydroxyl radicals , which have been suggested to play a role in eosinophil - mediated killing .
additionally , cyclooxygenase inhibitors and prostaglandin e2 , which have been found to inhibit nk cell cytotoxicity [ 47 , 48 ] and nf-b activation , were assessed . among the 3 hydroxyl radical scavengers used
similarly , only 1 ( salicylic acid ) of the 3 cycloxygenase inhibitors abrogated killing , while prostaglandin e2 failed to have any appreciable effect .
one study , however , described an effector mechanism by which il-15 stimulated cd8 ctl kill c. neoformans . in vitro studies
have previously shown that il-15 from c. neoformans - stimulated monocytes induced t cells to become anticryptococcal .
this led to studies showing that the anticryptococcal activity of il-15-stimulated cd8 t cells correlated with the level of granulysin expression . furthermore , both strontium treatment and sirna knockdown of granulysin abrogated anticryptococcal activity , directly implicating granulysin in the ctl response against c. neoformans .
perforin was not required , as neither concanamycin a nor egta treatment impaired anticryptococcal activity .
these results are in contrast to nk cells , which depend on perforin , but not granulysin for killing .  
these results were extended using cd8 t cells purified from pbmc that had been stimulated with c. neoformans mitogen ( cnm ) , a protein mitogen within the cryptococcal cell wall and membrane [ 52 , 53 ] .
priming of anticryptococcal activity and granulysin expression by stimulation with cnm was dependent on cd4 t cells .
stimulation of pbmc with cnm in the presence of anti - il-15 abrogated anticryptococcal activity and granulysin expression suggesting that the dependence on cd4 t cells was mediated through il-15 . however , in the absence of accessory cells , cd4 t cells were not sufficient to induce cd8 t cell anticryptococcal activity and granulysin expression . together
this data suggests that the proliferating cd4 t cells provide a retrograde stimulus to accessory cells that results in production of il-15 , which then primes cd8 t cell for granulysin expression and anticryptococcal activity ( figure 3 ) .
indeed , both cd4 and cd8 t cells in vivo have been found to be indispensable for an effective response to c. neoformans [ 5458 ] .   aside from their role in licensing accessory cells to prime cd8 ctl anticryptococcal activity ,
cd4 t cells have also been demonstrated to directly kill c. neoformans . upon stimulation with il-2
, cd4 t cells increased the expression of granulysin ( but not perforin ) which correlated with increased fungicidal activity .
both strontium treatment and granulysin sirna knockdown abrogated cd4 t cell killing of c. neoformans .
perforin involvement was excluded as neither concanamycin a nor perforin knockdown could interfere with anticryptococcal activity of cd4 t cells stimulated with both il-2 and anti - cd3 antibody ( which stimulated both perforin and granulysin expression ) . together
this data suggests that cd4ctl ( like cd8 ) directly kill c. neoformans through a granulysin - mediated mechanism .  
the high prevalence of serious cryptococcal infection in hiv - infected patients [ 60 , 61 ] may be at least partially due to a defect in this fungicidal activity .
indeed , cd4 t cells from these patients were found to exhibit defective killing of c. neoformans .
neither il-2 nor il-2 plus anti - cd3 stimulation could induce granulysin expression in these patients ' cells .
these results suggest that profound dysregulation of perforin and granulysin expression may account for the failure of cd4 t cells from hiv - infected patients to kill c. neoformans .
the signalling pathways regulating granulysin expression in cd8 and cd4 ctl are relatively unknown . in preparation for killing of fungi ,
expression of granulysin correlated with short - term ( 060 min ) and long - term ( 15 days ) phosphorylation of extracellular signal - regulated kinases ( erk)1/2 , p38 mitogen - activated protein ( map ) kinase , and p54 c - jun n - terminal kinases ( jnk ) as well as the transcription factor signal transducers and activators of transcription ( stat)5 . meanwhile , no phosphorylation of the human oncogene akt ( signifying phosphoinositide 3-kinase ( pi3k ) activation ) could be detected following continuous il-2 stimulation .
akt phosphorylation could only be detected when cd4 t cells were stimulated with il-2 for 35 days , rested ( 24 hours ) and restimulated with il-2 for 5 min , indicating that pi3k undergoes a transient activation after initial stimulation with il-2 .
continuous stimulation of cd4 t cells with il-2 was also found to increase expression of the il-2r and il-2r subunits of the il-2 receptor ( by day 3 ) followed by expression of granulysin ( on day 5 ) . pharmacological inhibition of janus kinase ( jak)3/stat5 or pi3k abrogated
both granulysin expression and anticryptococcal activity of il-2 stimulated cd4 t cells , which correlated with decreased expression of the il-2r subunits .
together this data suggests that granulysin expression requires acquisition of one or more of the il-2r subunits .
indeed , blockade of il-2r expression using anti - il-2r antibodies or sirna knockdown abrogated granulysin expression in il-2 stimulated cd4 t cells .
thus , il-2 ( and perhaps other t cell growth factors ) signals t cells to increase expression of il-2r ,   which is then available for signalling that is necessary for granulysin expression .  
previous studies have shown that cd4 t cells from hiv - infected patients exhibit dysregulated expression of perforin and granulysin .
comparison of il-2 signalling in cd4 t cells from healthy donors and patients infected with hiv revealed the underlying cause to be defective stat5 and pi3k signalling .
il-2 stimulation of cd4 t cells from hiv - infected patients failed to induce phosphorylation of stat5 and failed to increase expression of il-2r. furthermore , in contrast to cd4 t cells from healthy donors , il-2 failed to induce phosphorylation of akt .
together these results demonstrate a critical role for jak / stat and pi3k signalling in granulysin - mediated killing of c. neoformans by cd4 t cells .  
/ stat signalling was also found to be required for granulysin expression in cd8 t cells in response to il-15 and il-21 .
cd8 t cells stimulated with either il-15 or il-21 were found to increase granulysin expression , which correlated with increased phosphorylation of stat3 and stat5 .
il-15 was found to induce phosphorylation of both stat3 and stat5 , while il-21 only induced phosphorylation of stat3 . pharmacological inhibition and sirna knockdown of jak
/ stat signalling was found to abrogate granulysin expression by cd8 t cells in response to il-15 and il-21 .
together this data indicates that jak / stat signalling regulates granulysin expression by cd8 t cells in response to il-15 and il-21 .
consistent with previous studies conducted with cd4 t cells [ 59 , 62 ] , hiv - infected cd8 t cells exhibited reduced phosphorylation of stat3 , stat5 , and granulysin expression in response to il-15 and il-21 as compared to mock - infected cells .
it is clear that both cd4 and cd8ctl mediate direct killing of a wide range of bacterial , parasitic , and fungal pathogens .
several studies reported a correlation between the frequency [ 20 , 21 ] or intensity   of binding and killing .
moreover , microscopy [ 37 , 40 , 41 ] revealed direct ctl - microbe interactions .
finally , t cells separated from c. neoformans by a porous membrane were unable to mediate killing .
contact may induce release of cytotoxic products by the ctl or provide a microenvironment or concentration at which the cytotoxic product can be efficacious .
the latter may explain why supernatants from activated t cells were often unable to mediate killing [ 20 , 34 , 40 , 41 ] .
second , killing is mediated primarily through the granule - exocytosis pathway . in many experimental systems , pretreatment of ctl with strontium ( which depletes granule contents ) was found to abrogate killing [ 13 , 17 , 44 , 59 ] .
furthermore , granulysin , a granule constituent , has been implicated in the killing of several microbes [ 13 , 1517 , 33 , 44 , 59 ] .
third , killing ( at least of extracellular microbes ) is neither antigen - specific nor mhc - dependent .
nonspecific stimulation with t cell growth factors [ 34 , 35 , 39 , 40 , 44 , 59 ] or mitogenic lectins [ 20 , 21 ] was often sufficient to prime the effector cells for killing .   
while granulysin appears to be highly involved in killing , the role of perforin and other effector molecules is not as clear . in the case of extracellular pathogens
, perforin has not been found to play a role in killing [ 54 , 59 ] , although it plays a role in direct killing by nk cells [ 51 , 64 , 65 ] .
by contrast , killing of intracellular microbes requires perforin [ 13 , 27 ] , although some investigations have suggested a perforin - independent mechanism . to be sure , this topic demands the attention of future studies .  
jak / stat and pi3k signalling have been found to be essential in ctl killing of c. neoformans .
defective jak / stat and pi3k signalling in ctl from hiv - infected patients [ 62 , 63 ] may explain why these patients experience such a high incidence of severe cryptococcal infection .   during the process of evolution
, ctl have developed the ability to specifically recognize altered self through complex tcr - mhc interactions . as a consequence , they have become the epitome of specific cell - mediated immunity .
it is fascinating to now discover that during this process , ctl have , at the same time , preserved one of the most rudimentary immune functions of all , namely , the ability to directly recognize a microbe , bind , and without the help of antigen presenting cells or other effector cells kill the invading pathogen with competence and precision .
 cytotoxic t - lymphocytes ( ctl ) are famous for their ability to kill tumor , allogeneic and virus - infected cells . 
 however , an emerging literature has now demonstrated that ctl also possess the ability to directly recognize and kill bacteria , parasites , and fungi . here 
 , we review past and recent findings demonstrating the direct microbicidal activity of both cd4 + and cd8 + ctl against various microbial pathogens . 
 further , this review will outline what is known regarding the mechanisms of direct killing and their underlying signalling pathways . 
1. Introduction
2. Direct Killing of Bacteria by CTL
3. Direct Killing of Parasites by CTL
4. Direct Killing of Fungi by CTL
5. Summary